Columbia  ®nit)em'tp     . 

College  of  ^fjpsiitians!  anlJ  ^urseonjs 


Xibrarp 


(iH  Presented  by 

,  DR.  WILLIAM  J.  OIES^J^ 

;o  enrich  the  library  resources  '  |f 

avciiUble  to  holders 

OlES  FELLOWSHIP 

t>j  Biologic&l  Chemistry 


PRACTICAL 
PHYSIOLOGICAL   CHEMISTRY 

HAWK 


Absorption  Spectra. 


Oxyhaemoglobin. 


Haemoglobin. 


Carboxy- 
haemoglobin. 


Neutral  Met- 
haemoglobin. 


Alkaline  Met- 
haemoglobin. 


Alkali 
Haematin. 


Absorption  Spectra. 


Reduced  Alkali 
Haematin  or 
HaemochromoKen. 


Acid  Haematin  in 
ethereal  solution. 


Acid  Haemato- 
porphyrln. 


Alkaline 

Haematopor- 

phyrln. 


Urobilin  or  Hydro- 
bilirubin  In  acid 
solution. 


Urobilin  or  Hydro- 
bllirublivJn  alkaline 
solution  after  the 
addition  of  zinc 
chloride  solution. 


Blllcyanin  or 
Cholecyanin  in 
alkaline  solution. 


Digitized  by  tine  Internet  Archive 

in  2010  witii  funding  from 
Columbia  University  Libraries 


http://www.archive.org/details/practicalphysiol1912hawk 


PRACTICAL 


PHYSIOLOGICAL  CHEMISTRY 


A  BOOK  DESIGNED  FOR  USE  IN  COURSES  IN  PRACTIC 
PHYSIOLOGICAL   CHEMISTRY  IN  SCHOOLS 
OF  MEDICINE  AND  OF  SCIENCE 


BY 


PHILIP  B.  HAWK,  M.  S.,  Ph.  D. 

PROFESSOR   OF   PHYSIOLOGICAL  CHEMISTRY   AND   TOXICOLOGY   IN   THE 
JEFFERSON   MEDICAL  COLLEGE   OF   PHILADELPHIA 


FOURTH  EDITION,  REVISED  AND  ENLARGED 


WITH  TWO  FULL-PAGE  PLATES  OF  ABSORPTION  SPECTRA  IN  COLORS 
FOUR  ADDITIONAL  FULL-PAGE  COLOR  PLATES  AND  ONE 
HUNDRED  AND  THIRTY-SEVEN  FIGURES  OF  WHICH 
TWELVE  ARE  IN  COLORS 


PHILADELPHIA 

P.   BLAKISTON'S   SON   &   CO. 

1012   WALNUT   STREET 
1912 


First  Edition,  Copyright,  1907,  by  P.  Blakiston's  Son  &  Co. 
Second  Edition,  Copyright,  1909,  by  P.  Blakiston's  Son  &  Co. 

.Third  Edition,  Copyright,  iqio,  by  P.  Blakiston's  Son  &  Co. 
Fourth  Edition,  Copyright,  1912,  by  P.  Blakiston's  Son  Sz  Co. 


P 


THE. MAPLE. PRESS. TORK- PA 


THESE    PAGES   ARE 

AFFECTIONATELY    DEDICATED 

TO 

MY    MOTHER 


PREFACE  TO  FOURTH   EDITION. 

The  continued  rapid  development  of  the  many  phases  of  biochemistry, 
which  has  taken,  place  in  the  interval  of  two  years  since  the  last  edition 
of  this  volume  appeared  has  necessitated  a  rather  comprehensive  revision 
and  the  consequent  inclusion  of  considerable  new  matter.  The  main 
bulk  of  the  material  will  be  found  in  the  chapters  on  Enzymes,  Carbo- 
hydrates, Proteins,  Blood  and  Lymph,  Feces,  Putrefaction  and  Quanti- 
tative Analysis  of  Urine.  The  publishers  have  wisely  reduced  the 
marginal  space  of  the  page  thus  necessitating  but  slight  increase  in  the 
size  of  the  volume. 

The  author  wishes  to  express  his  gratitude  to  Professor  William  J. 
Gies,  Professor  Lafayette  B.  Mendel  and  Dr.  Thomas  B.  Osborne  for 
many  valuable  suggestions  for  the  betterment  of  this  revised  volume.  He 
is  also  under  obligations  to  Dr.  Martha  Tracy  and  Professors  Marshall 
P.  Cram,  Paul  E.  Howe,  E.  C.  L.  Miller,  Charles  J.  Robinson  and 
A.  P.  Sy  for  similar  offices  and  to  Messrs.  Olaf  Bergeim,  Lawrence  T. 
Fairhall,  Edwin  F.  Hirsch,  Melvin  A.  Saylor  and  Theodore  F.  Zucker 
for  assistance  in  the  verification  of  tests  and  methods,  the  translation  of 
papers,  the  sketching  of  crystals  and  the  reading  of  proof. 

The  author  would  be  grateful  if  those  using  the  volume  in  their 
classes  would  make  suggestions  regarding  insertions,  omissions  or 
corrections. 

Philip  B.  Hawk. 

Philadelphia. 


IIV 


PREFACE  TO  THIRD  EDITION. 

The  increasing  approval  with  which  this  volume  is  being  received  has 
rendered  necessary  the  preparation  of  a  new  edition,  although  the  period 
elapsing  since  the  last  edition  appeared  is  little  more  than  one  year. 
The  present  edition  has  been  brought  up  to  date  by  the  insertion  of 
various  additions  and  corrections  as  well  as  by  the  inclusion  of  a  number 
of  qualitative  tests  and  quantitative  methods.  Because  of  the  very  short 
intervening  period  since  the  last  edition  of  the  volume,  the  new  material 
inserted  is  rather  small  in  quantity  when  compared  with  that  incorporated 
at  the  previous  revision. 

The  author  wishes  to  thank  Dr.  W.  H.  Welker  and  Dr.  Croll  for 
permission  to  insert  unpublished  material. 

Philip  B.  Hawk. 

Urbana,  Illinois. 


UC 


PREFACE  TO  SECOND  EDITION. 

The  kind  fCccption  accorded  this  volume  by  the  instructors  in 
physiological  chemistry  in  the  United  States  and  Great  Britain  has 
made  the  preparation  of  a  new  edition  imperative,  notwithstanding 
the  fact  that  less  than  two  years  have  elapsed  since  the  former  edition 
appeared.  The  advance  and  development  made  in  the  field  of  physio- 
logical chemistry  during  this  period  have  been  both  rapid  and  impor- 
tant; conditions  which  would  of  themselves  have  necessitated  the  revision 
of  the  volume  at  an  early  date. 

The  book  has  been  thoroughly  revised  in  all  department  and  in 
part  rewritten,  the  system  of  spelling  officially  adopted  by  the  American 
Chemical  Society  having  been  followed  throughout  the  volume.  Besides 
introducing  many  new  qualitative  tests  and  quantitative  methods,  the 
author  has  added  a  chapter  on  "Enzymes  and  Their  Action"  and  has 
rewritten  the  two  chapters  on  Proteins.  The  term  "protein"  has  been 
substituted  for  "proteid"  and  the  classification  of  proteins  as  recently 
adopted  by  the  American  Physiological  Society  and  the  American  Society 
of  Biological  Chemists  has  been  introduced  and  is  followed  throughout 
the  text;  the  classification  adopted  by  the  Chemical  and  Physiological 
Societies  of  England  is  also  included. 

The  original  plan  of  the  book  has  been  adhered  to  with  the  excep- 
tion that  the  chapter  on  "Enzymes  and  Their  Action"  has  been  made 
Chapter  I  and  the  practical  work  upon  the  proteins  is  preceded  by  a 
chapter  giving  a  brief  discussion  of  protein  substances  from  the  stand- 
point of  their  decomposition  and  synthesis.  We  believe  that  the  student 
will  be  able  to  pursue  his  practical  work  more  intelligently  and  will 
derive  greater  benefit  therefrom  if  the  plan  of  instruction  as  suggested 
in  Chapters  IV  and  V  be  followed  in  the  presentation  of  the  subject 
of  "Proteins." 

The  author  wishes  to  express  his  thanks  to  all  those  who  so  kindly 
offered  suggestions  for  the  betterment  of  the  book.  He  is  particularly 
desirous  of  expressing  his  gratitude  to  Professor  Lafayette  B.  Mendel 
and  Dr.  Thomas  B.  Osborne  for  the  many  helpful  suggestions  they 
have  so  kindly  given  him.  His  thanks  are  also  due  Professor  C.  A. 
Herter,  Dr.  H.  D.  Dakin,  Dr.  S.  R.  Benedict,  and  Mr.  S.  C.  Clark 
for  permission  to  insert  unpublished  material,  to  Mr.  Paul  E.  Howe 

xi 


XU  PREFACE    TO    SECOND    EDITION. 

for  valuable  assistance  rendered  in  the  reading  of  proof  and  in  the  verifi- 
cation of  tests  and  methods,  and  to  Dr.  M.  E.  Rehfuss  for  assistance  in 
proof  reading. 

The  author  takes  this  opportunity  of  making  an  acknowledgment 
which  was  inadvertently  omitted  from  the  first  edition.  He  wishes  to 
express  his  obligation  to  the  laboratories  of  physiological  chemistry  at 
Yale  University  and  at  Columbia  University  (College  of  Physicians  and 
Surgeons)  in  the  latter  of  which  he  was  Assistant  to  Professor  W.  J. 
Gies  for  two  years.  The  courses  given  in  these  laboratories  formed  the 
basis  of  many  of  the  experiments  included  in  this  volume,  and  it  is  with 
feelings  of  deepest  gratitude  that  he  records  this  acknowledgment  of  the 
assistance  thus  rendered  by  those  in  charge  of  these  courses. 

Philip  B.  Hawk. 

Uebana,  Illinois. 


PREFACE  TO  FIRST  EDITION. 

The  plan  followed  in  the  presentation  of  the  subject  of  this  volume 
is  rather  different,  so  far  as  the  author  is  aware,  from  that  set  forth 
in  any  similar  volume.  This  plan,  however,  he  feels  to  be  a  logical 
one  and  has  followed  it  with  satisfactory  results  during  a  period  of 
three  years  in  his  own  classes  at  the  University  of  Pennsylvania.  The 
main  point  in  which  the  plan  of  the  author  differs  from  those  previously 
proposed  is  in  the  treatment  of  the  food  stuffs  and  their  digestion. 

In  Chapter  IV  the  "Decomposition  Products  of  Proteids"  has  been 
treated  although  it  is  impracticable  to  include  the  study  of  this  topic 
in  the  ordinary  course  in  practical  physiological  chemistry.  For  the 
specimens  of  the  decomposition  products,  the  crystalHne  forms  of  which 
are  reproduced  by  original  drawings  or  by  microphotographs,  the  author 
is  indebted  to  Dr.  Thomas  B.  Osborne  of  New  Haven,  Conn. 

Because  of  the  increasing  importance  attached  to  the  examination 
of  feces  for  the  purposes  of  diagnosis,  the  author  has  devoted  a  chapter 
to  this  subject.  He  feels  that  a  careful  study  of  this  topic  deserves 
to  be  included  in  the  courses  in  practical  physiological  chemistry,  of 
medical  schools  in  particular.  The  subject  of  solid  tissues  (Chapters 
XIII,  XIV  and  XV)  has  also  been  somewhat  more  fully  treated  than  has 
generally  been  customary  in  books  of  this  character. 

The  author  is  deeply  indebted  to  Professor  Lafayette  B.  Mendel, 
of  Yale  University,  for  his  careful  criticism  of  the  manuscript  and  to 
Professor  John  Marshall,  of  the  University  of  Pennsylvania,  for  his 
painstaking  revision  of  the  proof.  He  also  wishes  to  express  his  grati- 
tude to  Dr.  David  L.  Edsall  for  his  criticism  of  the  clinical  portion  of 
the  volume;  to  Dr.  Otto  Folin  for  suggestions  regarding  several  of  his 
quantitative  methods,  and  to  Mr.  John  T.  Thomson  for  assistance  in 
proof  reading. 

For  the  micro-photographs  of  oxyhaemoglobin  and  haemin  reproduced 
in  Chapter  XI  the  author  is  indebted  to  Professor  E.  T.  Reichert,  of  the 
University  of  Pennsylvania,  who,  in  collaboration  with  Professor  A.  P. 
Brown,  of  the  University  of  Pennsylvania,  is  making  a  very  extended 
investigation  into  the  crystalline  forms  of  biochemic  substances.  The 
micro-photograph  of  allantoin  was  kindly  furnished  by  Professor  Mendel. 

xiii 


XIV  PREFACE    TO    FIRST    EDITION. 

The  author  is  also  indebted  for  suggestions  and  assistance  received  from 
the  lectures  and  published  writings  of  numerous  authors  and  investigators. 
The  original  drawings  of  the  volume  were  made  by  Mr.  Louis 
Schmidt  whose  eminently  satisfactory  efforts  are  highly  appreciated  by 
the  author. 

Philip  B.  Hawk. 

Philadelphia. 


CONTENTS 


chapti:r  I. 

Page 

Enzymes  and  Their  Action i 

CHAPTER  II. 
Carbohydrates 25 

CHAPTER  III. 

Salivary  Digestion 59 

CHAPTER  IV. 

Proteins:  Their  Decomposition  AND  Synthesis 68 

CHAPTER  V. 
Proteins:  Their  Classification  and  Properties 92 

CHAPTER  VI. 
Gastric  Digestion 124 

CHAPTER  VII. 
Fats 139 

CHAPTER  VIII. 

Pancreatic  Digestion ,    .   148 

CHAPTER  IX. 
Bile 158 

CHAPTER  X. 
Putrefaction  Products 169 

CHAPTER  XI. 

Feces 178 

CHAPTER.XII. 

Blood  and  Lymph 194 

XV 


XVI  CONTENTS. 

CHAPTER  XIII. 

Pagb 
^JlLK        235 

CHAPTER  XIV. 
Epithelial  and  Connective  Tissues 245 

CHAPTER  XV. 
Muscular  Tissue 254 

CHAPTER  X\T. 
Nervous  Tissue 268 

CHAPTER  XVII. 

Urine:  Geneila.l  ChlVracteristics  of  Normal  and  Pathological  Urine  274 

CH.APTER  XVIII. 
Urine:  Physiological  Constituents 283 

CHAPTER  XIX. 

Urine:  Pathological  Constituents     323 

CHAPTER  XX. 

Urine:  ORGAN^ZED  and  Unorganized  Sediments 361 

CHAPTER  XXI. 
Urine:  Calculi 379 

CHAPTER  XXII. 
Urine:  Quantitative  Analysis 383 

CHAPTER  XXIII. 

Quantitative  Analysis  of  Milk,  Gastric  Juice  and  Blood 435 

Appendix     443 

Index 455 


LIST  OF  ILLUSTRATIONS 


Plate 

I.  Absorption  Spectra    "1                                                                     c        •  • 

II.  Absorption  Spectra    / ^ 

III.  Osazones Opposite  page  28 

IV.  Normal  Erythrocytes  and  Leucocytes Opposite  page  196 

V.  Uric  Acid  Crystals Opposite  page  291 

VI.  Ammonium  Urate Opposite  page  365 

Figure  Page 

1.  Apparatus  for  Quantitative  Determination  <jr  Catalase 24 

2.  Dialyzing  Apparatus  for  Students'  Use 30 

3.  Einhorn  Saccharometer o6 

4.  One  Form  of  Laurent  Polariscope 37 

5.  Diagrammatic  Representation  of  the  Course  of  the  Light  through  the 

Laurent  Polariscope ■jg 

6.  Polariscope  (Schmidt  and  Hansch  Model)      38 

7.  Iodoform 4y 

8.  Potato  Starch 4p 

9.  Bean  Starch ^p 

10.  Arrowroot  Starch ^g 

11.  Rye  Starch ^g 

12.  Barley  Starch ^g 

13.  Oat  Starch ^p 

14.  Buckwheat  Starch 40 

15.  Maize  Starch ^g 

16.  Rice  Starch 4^ 

17.  Pea  Starch 4g 

18.  Wheat  Starch 4q 

19.  Microscopical  Constituents  of  Saliva 63 

20.  Glycocoll  Ester  Hydrochloride y-j 

21.  Serine 78 

22.  Phenylalanine jg 

23.  Fischer  Apparatus 80 

24.  Tyrosine 81 

25.  Cystine      81 

26.  Histidine  Dichloride 8? 

27.  Leucine 85 

28.  Lysine  Picrate      86 

29.  Aspartic  .\cid 86 

xvii 


XVlll  LIST    OF    ILLUSTEATICNS. 

Figure  Page 

30.  Glutamic  Acid 87 

31.  Laevo- a-Proline 88 

32.  Copper  Salt  of  Proline 89 

33.  Coagulation  Temperature  Apparatus 107 

34.  Edestin no 

35.  Excelsin,  the  Protein  of  the  Brazil  Nut no 

36.  Beef  Fat 139 

37.  Mutton  Fat 142 

38.  Pork  Fat • 144 

39.  Palmitic  Acid 145 

40.  Melting-Point  Apparatus 146 

41.  Bile  Salts 160 

42.  Bilirubin  (Hsematoidin)      i6r 

43.  Cholesterol 166 

44.  Taurine 167 

'45.   Glycocoll 167 

46.  Ammonium  Chloride      175 

47.  Microscopical  Constituents  of  Feces 178 

48.  Hsematoidin  Crystals  from  Acholic  Stools 179 

49.  Charcot-Leyden  Crystals ■   .    181 

50.  Boas'  Sieve 184 

51.  Oxyhsemoglobin  Crystals  from  Blood  of  the  Guinea  Pig      198 

52.  Oxyhsemoglobin  Crystals  from  Blood  of  the  Rat 198 

53.  Oxy  haemoglobin  Crystals  from  Blood  of  the  Horse 199 

54.  Oxyhaemoglobin  Crystals  from  Blood  of  the  Squirrel 199 

55.  Oxy haemoglobin  Crystals  from  Blood  of  the  Dog 200 

56.  Oxyhaemoglobin  Crystals  from  Blood  of  the  Cat 200 

57.  Oxyhaemoglobin  Crystals  from  Blood  of  the  Necturus 201 

58.  Effect  of  Water  on  Erythrocytes 208 

59.  Haemin  Crystals  from  Human  Blood 211 

60.  H^min  Crystals  from  Sheep  Blood .211 

61.  Sodium  Chloride 213 

62.  Direct-vision  Spectroscope 216 

63.  Angular- vi.sion  Spectroscope  Arranged  for  Absorption  Analysis      .    .217 

64.  Diagram  of  Angular-\dsion  Spectroscope 217 

65.  Fleischl's  Haemometer 220 

66.  Pipette  of  Fleischl's  Haemometer 221 

67.  Colored  Glass  Wedge  of  Fleischl's  Haemometer 221 

68.  Dare's  Haemoglobinometer 222 

69.  Horizontal  Section  of  Dare's  Haemoglobinometer 223 

70.  Method  of  Filling  the  Capillary  Observation  Cell  of  Dare's  Haemo- 

globinometer     223 

71.  Thoma-Zeiss  Counting  Chamber 224 

72.  Thoma-Zeiss  Capillary  Pipettes 225 


LIST    Ol     ILLUSTRATIONS.  xix 

FicLRE  Page 

73.  Ordinary  Ruling  of  Thoma-Zeiss  Counting  Chamber 226 

74.  Zappert's  Modified  Ruling  of  Thoma-Zeiss  Counting  Chamber.    .    .  227 

75.  Biirker's  Pipettes,  Mixing  Flasks,  and  Counting  Chamber 229 

76.  Ruling  of  Biirkcr  Counting  Chamber      232 

77.  Schema 232 

78.  Burker  Counting  Chamber 233 

79.  Normal  Milk  and  Colostrum 236 

80.  Lactose 238 

8r.   Calcium  Phosphate 242 

82.  Creatine 257 

83.  Xanthine 258 

84.  Hypoxanthine  Silver  Nitrate 265 

85.  Xanthine  Silver  Nitrate      266 

86.  Deposit  in  Ammoniacal  Fermentation 277 

87.  Deposit  in  Acid  Fermentation 277 

88.  Urinometer  and  Cylinder 278 

89.  Beckmann-Heidenhain  Freezing-point  .Apparatus 280 

90.  Urea 285 

91.  Urea  Nitrate 287 

92.  Melting-point  Tubes  Fastened  to  Bulb  of  Thermometer 288 

93.  Urea  Oxalate 289 

94.  Pure  Uric  Acid 293 

95.  Creatinine 295 

96.  Creatinine-Zinc  Chloride 296 

97.  Hippuric  Acid      300 

98.  AUantoin  from  Cat's  Urine 304 

99.  Benzoic  Acid 308 

100.  Calcium  Sulphate 316 

loi.  "Triple  Phosphate" 310 

102.  The  Purdy  Electric  Centrifuge 361 

103.  Sediment  Tube  for  the  Purdy  Electric  Centrifuge 361 

104.  Calcium  Oxalate 363 

105.  Calcium  Carbonate 363 

106.  Various  Forms  of  Uric  Acid 365 

107.  Acid  Sodium  Urate 366 

108.  Cystine 366 

109.  Crystals  of  Impure  Leucine 367 

no.  Epithelium  from  Different  Areas  of  the  Urinary  Tract 370 

111.  Pus  Corpuscles 3yi 

112.  Hyaline  Casts 3-2 

113.  Granular  Casts ^-jj 

114.  Granular  Casts 374 

115.  Epithelial  Casts 374 

116.  Blood,  Pus,  Hyaline  and  Epithelial  Casts 374 


XX  LIST    OF    ILLUSTRATIONS. 

FifiuRE  Page 

117.  Fatty  Casts 375 

118.  Fatty  and  Waxy  Casts 375 

119.  Cylindroids 376 

120.  Crenated  Erythrocytes 377 

121.  Human  Spermatozoa      377 

122.  Esbach's  Albuminometer 384 

123.  Marshall's  Urea  Apparatus 392 

124.  Hiifner's  Urea  Apparatus 394 

125.  Doremus-Hinds  Ureometer 395 

126.  Folin's  Urea  Apparatus      396 

127.  Folin's  Ammonia  Apparatus 399 

128.  Folin  Improved  Absorption  Tube 400 

129.  130.  Forms  of  Apparatus  used  in  Methods  of  Folin  and  Associates  for 

Determination  of  Total  Nitrogen,  Urea  and  Ammonia 403 

'131.  Van  Slyke's  Amino-nitrogen  Apparatus 405 

132.  Berthelot-Atwater  Bomb  Calorimeter      411 

133.  Hall's  Purinometer 431 

134.  Centrifuge  Tube  used  in  Babcock  Fat  Method 435 

135.  Croll's  Fat  Apparatus 436 

136.  Soxhlet  Apparatus 437 

137.  Feser's  Lactoscope 438 


PHYSIOLOGICAL  CHEMISTRY. 


CHAPTER  I. 

-  ENZYMES  AND  THEIR  ACTION. 

According  to  the  old  classification  ferments  were  divided  into  two 
classes,  the  organized  ferments  and  the  unorganized  ferments.  As  organized 
ferments  or  true  ferments  there  w^ere  grouped  such  substances  as  yeast 
and  certain  bacteria  which  were  supposed  to  act  by  virtue  of  vital  processes, 
whereas  the  unorganized  ferments  included  salivary  amylase  (ptyalin), 
gastric  protease  (pepsin),  pancreatic  protease  (trypsin),  etc.,  which  were 
described  as  "non-li^ing  unorganized  substances  of  a  chemical  nature." 
Kiihne  designated  this  latter  class  of  substances  as  enzymes  {iv  ^vfirj-m  yeast). 
This  division  into  organized  ferments  (true  ferments)  and  unorganized 
ferments  (enzymes)  was  generally  accepted  and  was  practically  unc^ues- 
tioned  until  Buchner  overthrew  it  in  the  year  1897  by  his  epoch-making 
investigations  on  zymase.  Previous  to  this  time  many  writers  had  ex- 
pressed the  opinion  that  the  action  of  the  ferment  organisms  was  similar 
to  that  of  the  unorganized  ferments  or  enzymes  and  that  therefore  the 
activity  of  the  former  was  possibly  due  to  the  production  of  a  substance 
in  the  cell,  which  was  in  nature  similar  to  an  enzyme.  Investigation 
after  investigation,  however,  failed  to  isolate  any  such  principle  from  an 
active  cell  and  the  exponents  of  the  "vital"  theory  became  strengthened 
in  their  belief  that  certain  fermentative  processes  brought  about  by  living 
cells  could  not  occur  apart  from  the  biological  acti\-ity  of  such  cells.  How- 
ever, as  early  as  1858,  Traube  had  enunciated,  in  substance,  the  principles 
which  were  destined  to  be  fundamental  in  our  modern  theory  of  fermenta- 
tion. He  expressed  the  belief  that  the  yeast  cell  produced  a  product  in 
its  metabolic  actiA-ities  which  had  the  property  of  reacting  with  sugar  with 
the  production  of  carbon  dioxide  and  alcohol,  and  further  that  this 
reaction  between  the  product  of  the  metabolism  of  the  yeast  cell  and  the 
sugar  occurred  without  aid  from  the  original  cell.  It  was  not  until  1897, 
however,  that  this  theory  was  placed  upon  a  firm  experimental  basis. 
This  was  brought  about  through  the  efforts  of  Buchner  who  succeeded  in 
isolating  from  the  living  yeast  cells  a  substance  (zymase)  which,  when 
freed  from  the  last  trace  of  organized  cellular  material,  was  able  to  bring 
about  the  identical  fermentative  processes,  which,  up  to  this  time,  had 
been  deemed  possible  only  in  the  presence  of  the  active,  living  yeast  cell. 
Buchner's  manipulation  of  the  yeast  cells  consisted  in  first  grind- 


2  PHYSIOLOGICAL    CHEMISTRY. 

ing  them  with  sand  and  infusorial  earth,  after  which  the  finely  divided 
material  was  subjected  to  great  pressure  (300  atmospheres)  and  yielded 
a  liquid  which  possessed  the  fermentative  activity  of  the  unchanged 
yeast  cell/  This  liquid  contained  zymase,  the  principal  enzyme  of 
the  yeast  cell.  Later  the  lactic-acid-  and  acetic-acid-producing  bac- 
teria were  subjected  by  Buchner  to  treatment  similar  to  that- accorded 
the  yeast  cells,  and  the  active  intracellular  enzymes  were  obtained.  Many 
other  instances  are  on  record  in  which  a  soluble,  active  agent  has  been 
isolated  from  ferment  cells,  with  the  result  that  it  is  pretty  well  estab- 
lished that  all  the  so-called  organized  ferments  elaborate  substances  of 
this  character.  Therefore,  basing  our  definition  on  the  work  of  Buchner 
and  others  we  may  define  an  enzyme  as  an  unorganized,  soluble  ferment, 
which  is  elaborated  by  an  animal  or  vegetable  cell  and  whose  activity  is 
entirely  independent  of  any  of  the  life  processes  of  such  a  cell.  According 
to  this  definition  the  enzyme  zymase  elaborated  by  the  yeast  cell  is  entirely 
comparable  to  the  enzyme  pepsin  elaborated  by  the  cells  of  the  stomach 
mucosa.  One  is  derived  from  a  vegetable  cell,  the  other  from  an  animal 
cell,  yet  the  activity  of  neither  is  dependent  upon  the  integrity  of  the  cell. 

Enzymes  act  by  catalysis  and  hence  may  be  termed  catalyzers  or 
catalysts.  A  simple  rough  definition  of  a  catalyzer  is  "a  substance 
which  alters  the  velocity  of  a  chemical  reaction  mthout  undergoing 
any  apparent  physical  or  chemical  change  itself  and  without  becoming 
a  part  of  the  product  formed."  It  is  a  well-known  fact  that  the  velocity 
of  the  greater  number  of  chemical  reactions  may  be  changed  through 
the  presence  of  some  catalyzer.  For  example,  take  the  case  of  hydro- 
gen peroxide.  It  spontaneously  decomposes  slowly  into  the  water  and 
oxygen.  In  the  presence  of  colloidal  platinum,^  however,  the  decom- 
position is  much  accelerated  and  ceases  only  when  the  destruction  of 
the  hydrogen  peroxide  is  complete.  Without  multiplying  instances, 
suffice  it  to  say  that  there  is  an  analogy  between  inorganic  catalyzers 
and  enzymes,  the  main  point  of  difference  between  the  enzymes  and 
most  of  the  inorganic  cata  yzers  being  that  the  enzymes  are  colloids.^ 

Inasmuch  as  each  of  the  enzymes  has  an  action  which  is  more  or  less 
specific  in  character,  and  since  it  is  a  fairly  simple  matter,  ordinarily,  to 
determine  the  character  of  that  action,  the  classification  of  the  enzymes 
is  not  attended  with  very  great  difficulties.  They  are  ordinarily  classi- 
fied according  to  the  nature  of  the  substrate*  or  according  to  the  type 

'  In  later  investigations  the  process  was  improved  by  freezing  the  ground  cells  with  liquid 
air  and  finely  fiulverizing  them  before  applying  the  pressure. 

^  Produced  by  the  passage  of  electric  sparks  between  two  platinum  terminals  immersed 
in  distilled  water,  thus  liberating  ultra-microscopic  particles. 

'  Bredig  has  been  able  to  obtain  certain  inorganic  catalyzers  in  colloidal  solution.     These 
he  calls  "  inorganic  enzymes." 

*  Substance  acted  upon.     See  Lippmann:  Ber.  d.  Deulsch.  Chem.  Ces.,  36,  331,  1903. 


ENZYMES   AND    THEIR   ACTION.  3 

of  reaction  they  bring  about.  Thus  we  have  various  classes  of  enzymes, 
such  as  amylolytic,^  proteolytic,  lipolytic,  glycolytic,  uricolytic,  autolytic, 
oxidizing,  reducing,  inverting,  protein-coagulating,  deamidizing,  etc.  In 
every  instance  the  class  name  indicates  the  individual  type  of  enzy- 
matic activity  which  the  enzymes  included  in  that  class  are  capable  of 
accomplishing.  For  example,  amylolytic  enzymes  facilitate  the  hydro- 
lysis of  starch  (amylum)  and  related  substances,  lipolytic  enzymes 
facilitate  the  hydrolysis  of  fats  (Aittos),  whereas  through  the  agency  of 
uricolytic  enzymes  uric  acid  is  broken  down.  There  is  a  tendency, 
at  the  present  time,  to  harmonize  the  nomenclature  of  the  enzymes  by 
the  use  of  the  termination,  -ase.  According  to  this  system  of  nomen- 
clature, all  starch-transforming  enzymes,  or  so-called  amylolytic  en- 
zymes, are  called  amylases,  all  fat-splitting  enzymes  are  called  lipases, 
etc.  Thus  ptyalin  the  amylolytic  enzyme  of  the  saliva,  would  be 
termed  salivary  amylase  in  order  to  distinguish  it  from  pancreatic  amy- 
lase (amylopsin)  and  vegetable  amylases  (diastase,  etc.).  According 
to  the  same  system,  the  fat-splitting  enzyme  of  the  gastric  juice  would 
be  termed  gastric  lipase  to  differentiate  it  from  pancreatic  lipase  (steap- 
sin),  the  fat-splitting  enzyme  of  the  pancreatic  juice. 

Euler^  claims  that  enzymatic  cleavage  and  synthesis  are  often  brought 
about  by  two  different  components  of  an  enzyme  preparation.  He 
would  indicate  this  fact  by  giving  the  termination  -ese  to  those  enzymes 
exerting  a  synthetic  function.  For  example,  the  enzyme  which  catalyzes 
the  formation  of  nitriles  Euler  would  call  miriXese  in  distinction  from  nitril- 
ase  which  splits  nitriles.  He  would  further  designate  as  phosphatase 
the  enzyme  which  builds  up  phosphoric  acid  esters  of  carbohydrates  in 
distinction  from  phosphatase  which  causes  their  cleavage.  In  the  same 
way  he  would  differentiate  the  lipolytic  enzymes  into  lipases  and  lipeses. 

Our  knowledge  regarding  the  distribution  of  enzymes  has  been 
wonderfully  broadened  in  recent  years.  Up  to  within  a  few  years, 
the  real  scientific  information  as  to  the  enzymes  of  the  animal  organism, 
for  example,  was  limited,  in  the  main,  to  a  rather  crude  understanding 
of  the  enzymes  intimately  connected  with  the  main  digestive  func- 
tions of  the  organism.  We  now  have  occasion  to  believe  that  enzymes 
arc  doubtless  present  in  every  animal  cell  and  are  actively  associated 
with  all  vital  phenomena.  As  a  preeminent  example  of  such  cellular 
activity  may  be  cited  the  liver  cell  with  its  reputed  complement  of  15- 
20  or  more  enzymes. 

'Armstrong  suggests  the  use  of  the  termination  "clastic"  instead  of  "lytic."  He  calls 
attention  to  the  fact  that  amylolytic,  in  analogy  with  electrolytic,  means  "decomposition  by 
means  of  starch"  and  is  therefore  a  misnomer.  He  suggests  the  use  of  amyloclastic,  proteo- 
clastic,  etc. 

*  Euler:  Zeitschrift  fiir  physiologische  chemie,  74,  13,  1911. 


PHYSIOLOGICAL    CHEMISTRY. 


A  list  of  the  more  important  enzymes  together  with  their  class,  dis- 
tribution, substrate  and  end-products  is  given  below. 


Name. 


CLASSIFICATION  OF  ENZYMES. 
Class.  i        Distribution.  Substrate. 


End-products. 


Adenase Deamidizing \  Animal  tissues 


Adenine Hypoxanthine. 


Atnylases 

(a)  Pancreatic . . . 
(amylopsin) 

(b)  Salivary 

(ptyalin) 

(c)  Vegetable.  . . 
(diastase) 


Amylolytic. 


Pancreatic  juice..  .  .    Starch,  dextrine,     Malsose. 

etc.  _  I 

Saliva .  .■ '  Starch,  dex  trine,  ,  Maltose. 

etc. 
Plant  tissues '  Starch,  dex  trine,  i  Maltose. 

etc.  ! 


Arginase . 


Argenine-split- 
ting. 


Mucosa  of  intes- 
tine and  in  paren- 
chyma of  liver, 
kidney,  spleen, 
etc. 


Arginine . 


Ornithine  and  urea. 


Bromelin 

Proteolytic 

Pineapple 

....    Proteins 

Proteoses,  peptones, 
etc. 

Carboxylase 

Decarboxylizing. 

Yeast 

COOH  group  of  ah- 

phatic  acids. 

Carbon  dioxide. 

Catalase 

Oxidizing 

Tissues 

.....  Peroxides 

Oxygen,  water. 

Emulsin      (synap- 
tase). 

Glucoside-split- 
ting. 

Plants 

.  .  .  .  i  Amygdalin,  etc ...  . 

Glucose,  etc. 

Enterokinase Activating. 


Intestinal      epithe-     Trypsinogen. 
lium. 


Trypsin. 


Erepsin  (protease) . !  Proteoljrtic . 


Glycogenase . 


Glycogen-s  p  1  i  t- 
ting. 


Intestinal     mucosa     Proteoses,  peptones, 
of  man  and  dogs.       peptides,        and 
Animal  and  vege-       casein, 
table   tissues,   and 
pancreatic  juice. 


Simple  cleavage 
products,  such  as 
amino  acids. 


Liver,  intestinal     Glycogen, 
mucosa  (?),  mus- 
cles (?). 


Maltose  and  dextrin 
(dextrose  ?) . 


Glycolytic  enzymes  .    Glycolytic. 


Blood  and  various .  .    Sugar. 
organs. 


Lactic  acid,  alcohol, 
carbon  dioxide  and 
water. 


Guanase Deamidizing. 


Animal  tissues Guanine Xanthine. 


Laccase Oxidizing. 


Sap  of  lac  tree  and 
other  saps  and  • 
juices;  fungi;  gum 
arable,  etc. 


Polyhydric  p-phe- 
nols  such  as  hy- 
droquinone  and 
pyrogallol. 


Oxidation  products. 


Lactase . 


Lactose-splitting.    Intestinal  juice  and 
mucosa. 


Lactose. 


Dextrose  and  galac- 
tose. 


Lipases . 

(a)  Pancreatic. 
(steapsin) 

(b)  Gastric.  .  . 


(c)   Vegetable . 


Lipolytic . 


Pancreatic  juice. 
Gastric  juice. . . . 
Plant  tissues .... 


Neutral  fats. 
Neutral  fats. 
Neutral  fats. 


(d)  Animal. 


Animal  tissues Neutral  fats. 


Fatty    acid    and 

alcohol. 
Fatty    acid    and 

alcohol. 
Fatty    acid    and 

alcohol. 

Fatty    acid    and 
alcohol. 


Maltose '  Maltose-splitting. 


Blood  serum,  liver.     Maltose, 
saliva,   pancreatic 
and  intestinal | 
juice  and  lymph.      ' 


Dextrose. 


Nuclease. 


Nucleoprotein   Tissues Nucleoprotein. 

hydrolyzing. 


Purine  bases. 


Oxidases. 


Oxidizing . 


Plant    and    animal     Various  tissue  con-     Oxidation  products, 
tissues.  stituents. 


Pancreatic  rennin.  .    Coagulating Pancreatic  juice. 


Caseinogen I  Casein. 


ENZYMES   AND    THEIR   ACTION. 
CLASSIFICATION  OF  ENZYMES.— Contmued. 

Name.         |  Class.  j        Distribution.        [  Substrate.  End-products. 


Papain  (papayotin) ,  Proteolytic [  Pawpaw '  Proteins Proteoses,  peptones, 

I  etc. 

^ 1 j ^ 

Pepsin  (pepsase  or  j  Proteolytic Gastric  juice Proteins Proteoses,  peptones 

acid -proteinase).   ;  and  peptides. 

Peroxidases Oxidizing Plant    and    animal     Peroxides,    or    hy-     Oxidation  products. 

^  tissues.  droperoxides    and 

]  carries  oxygen  to 

tissue  constitu- 
ents. 


Phytase 

Phytin-splitting. . 

Rice  bran 

Phytin 

.  .  .  .  Inosite  and  phos- 
phoric acid. 

Protease  (erepsin) . . 

Proteolytic 

Kachree  gourd 

Proteins 

.  .  .  .  Proteoses,  peptones, 
peptides,  etc. 

Rennin     (rennase 
or  caseinase.) 

Coagulating 

Gastric    and    pan- 
creatic juices. 

Caseinogen .... 

.  .  .  .    Casein. 

Sucrose    (invertase 
or  invertin). 

Inverting 

Mucosa    and    juice 
of  the  intestine. 

Sucrose 

.  .  .  .    Dextrose    and    Lse- 
vulose      (invert- 
sugar)  . 

Thrombin 

Coagulating 

Blood 

Fibrinogen 

.  .  .  .    Fibrin. 

Trypsin    (trypsase 
or   alkali-protein- 
ase.) 

Proteolytic 

Pancreatic  juice..  .  . 

Proteins 

,  .  .  .  Proteoses,  peptones, 
peptides  and  ami- 
no acids. 

Tyrosinase Oxidizing Plant    and    animal     Tyrosine ,  Homogentisic    acid, 

tissues.  etc. 


Urease Urea-splitting. . . .    Micrococcus   ureae. .    Urea Carbon  dioxide  and 

1  ammonia. 


Uncase  (uricolytic  ,  Uric      acid-split-  j  Tissues Uric  acid AUantoin,  urea,  gly- 

enzyme).  ting.  cocoU  and  glyoxy- 

lic  acid  (?}. 


Xantho-oxidase. . . 

.    Oxidizing 

Tissues 

.    Xanthine   and 
poxanthine. 

hy- 

Uric  acid. 

Zymase 

.    S  u  g  a  r-ferment- 
ing. 

Yeast 

.    Sugar 

Alcohol  and  carbon 
dioxide. 

Inulase 

.    Hydrolytic 

Plants  and  fungi .  . 

Inulin 

Laevulose. 

Rhamnase 

.    Hydrolytic 

Fungi 

.    Rhamnose. 

Trehalase 

.     Hydrolytic 

Fungi 

.    Trehalose. 

1 

In  text-book  discussions  of  the  enzymes  it  is  customary  to  say  that 
very  little  is  known  regarding  the  chemical  characteristics  of  these  sub- 
stances since  no  member  of  the  enzyme  group  has,  up  to  the  present 
time,  been  prepared  in  an  absolutely  pure  condition.  Apparently,  how- 
ever, from  the  nature  of  the  facts  in  the  case,  it  would  be  much  more 
accurate  to  say  that  we  absolutely  do  not  know  whether  a  specific  enzyme 
has,  or  has  not,  been  prepared  in  a  pure  state.  (Some  authors,  like 
Arthus,  have  assumed  that  enzymes  are  not  chemical  individuals,  but 
properties  conferred  upon  bodies.)  The  enzymes  are  very  difficult  to 
prepare  in  anything  like  a  condition  approximating  purity,  since  they 
are  very  prone  to  change  their  nature  during  the  process  by  which  the 
investigator  is  attempting  to  isolate  them.  For  this  reason  we  have 
absolutely  no  proof  that  the  final  product  obtained  is,  or  is  not,  in  the 


t)  PHYSIOLOGICAL    CHEMISTRY. 

same  state  of  purity  it  possessed  in  the  original  cell.  Some  of  the  en- 
zymes are  more  or  less  closely  associated  with  the  proteins  from  the  fact 
that  they  are  both  formed  in  every  cell  as  the  result  of  the  cellular  ac- 
tiWty,  both  may  be  removed  from  solution  by  "salting-out,"  both  are 
for  the  most  part  non-diffusible  and  are  probably  very  similar  as  re- 
gards elementary  composition.  Hence  in  the  preparation  of  some 
enzymes  it  is  extremely  difficult  to  make  an  absolute  separation  from 
the  protein.*  Under  certain  conditions  enzymes  are  readily  adsorbed 
by  shredded  protein  material,  such  as  fibrin,  and  may  successfully 
resist  the  most  prolonged  attempts  at  washing  them  free.  We  may 
summarize  some  of  the  properties  of  the  great  body  of  enzymes  as  fol- 
lows: Enzymes  are  soluble  in  dilute  glycerol,  sodium  chloride  solu- 
tion, dilute  alcohol  and  water,  and  precipitable  by  ammonium  sulphate 
and  strong  alcohol.  Their  presence  may  be  proven  from  the  nature 
of  the  end-products  of  their  action  and  not  through  the  agency  of  any 
chemical  test.  They  are  colloidal  and  non- diffusible,  and  occur  closely 
associated  with  protein  material  with  which  they  possess  many  proper- 
ties in  common.  Each  enzyme  shows  the  greatest  activity  at  a  certain 
temperature  called  the  optimum  temperature;  there  is  also  a  minimum 
and  a  maximum  temperature  for  each  specific  enzyme.  Their  action 
is  inhibited  by  sufficiently  lowering  the  temperature,  and  the  enzyme,  if 
in  solution,  is  entirely  destroyed  by  subjecting  it  to  a  temperature  of 
ioo°  C.  The  best  known  enzymes,  whether  derived  from  warm-blooded 
or  cold-blooded  animals,  are  most  active  between  35°-45°  C.  The 
nature  of  the  surrounding  media  alters  the  velocity  of  the  enzymatic 
action,  some  enzymes  being  more  active  in  acid  solution  whereas  others 
require  an  alkaline  fluid. 

Many  of  the  more  important  enzymes  do  not  occur  preformed 
within  the  cell,  but  are  present  in  the  form  of  a  zymogen  or  mother- 
substance.  In  order  to  yield  the  active  enzyme  this  zymogen  must  be 
transformed  in  a  certain  specific  manner  and  by  a  certain  specific  sub- 
stance. This  transformation  of  the  inactive  zym.ogen  into  the  active 
enzyme  is  termed  activation.  For  instance,  the  zymogen  of  the  enzyme 
pepsin  of  the  gastric  juice,  termed  pepsinogen,  is  activated  by  the  hydro- 
chloric acid  secreted  by  the  gastric  cells  (see  p.  127),  whereas  the  acti- 
vation of  the  trypsinogen  of  the  pancreatic  juice  is  brought  about  by  a 
substance  termed  enterokinase^  (see  p.  15).  These  are  examples  of 
many  well-known  activation  processes  going  on  continually  within  the 
animal  organism.  The  agency  which  is  instrumental  in  activating  a 
zymogen  is  generally  termed  a  zymo-exciter  or  a  kinase.     In  the  cases 

'  Others  seem  to  be  like  the  substrate  on  which  they  act,  e.  g.,  carbohydrate. 

^  According  to  Delezenne,  trypsinogen  may  be  rapidly  activated  by  soluble  calcium  salts 


ENZYMES   AND    THEIR  ACTION.  7 

cited  hydrochloric  acid  would   be  termed  a  zymo-exciter  and  entero- 
kinase  would  be  termed  a  kinase. 

After  filterintT  yeast  juice,  prej)ared  by  the  Buchner  process  (see  p. 
i),  through  a  Martin  gelatin  filter,  Harden  and  Young  showed  that 
the  colloids  left  behind  and  the  filtrate  were  both  inactive  fermenta- 
tively.  Upon  treating  the  colloid  material  (enzyme)  with  some  of  the 
filtrate,  however,  the  mixture  was  shown  to  be  able  to  bring  about  pro 
nounced  fermentation.  It  is  believed  that  a  co-enzyme  present  in  the 
filtrate  was  the  eflScient  agent  in  the  transformation  of  the  inactive 
enzyme.  It  is  necessary  to  make  frequent  renewals  of  the  co-enzyme 
in  order  to  maintain  continuous  fermentation.  It  was  further  shown 
that  this  co-enzyme,  in  addition  to  being  diffusible,  was  not  destroyed 
by  boiling  and  that  it  disappeared  from  yeast  juice  when  this  latter 
w^as  fermented  or  allowed  to  undergo  autolysis.  The  exact  nature  of 
this  co-enzyme  of  zymase  is  unknown.  The  co-enzyme  action,  in  this 
case,  is  probably  dependent  upon  the  presence  of  two  individual  agencies, 
one  of  which  is  phosphates. 

It  has  been  shown  by  Loevenhart  that  the  property  of  acting  as  a 
pancreatic  lipase  co-enzyme  is  vested  in  bile  salts,  and  Magnus  has 
further  shown  that  the  synthetic  salts  are  as  efficient  in  this  regard  as 
the  natural  ones.  A  few  other  instances  of  co-enzyme  demonstrations 
have  been  reported. 

Electrolytes  are  very  important  factors  in  facilitating,  or  inhibiting 
enzyme  action.  ^  For  example,  magnesium  hydroxide  inhibits  the  action 
of  salivary  amylase"  whereas  the  CI  ion  facilitates  the  action  of  this  and 
other  amylases.^  In  fact  Bierry^  has  very  recently  gone  so  far  as  to  assert 
that  the  presence  of  the  CI  or  Br  ion  is  ''absolutely  essential  to  the  activity 
of  pancreatic  amylase." 

The  so-called  "specificity"  of  enzyme  action  is  an  interesting  and 
important  fact.  That  enzymes  are  very  specific  as  to  the  character  of 
the  substrate,  or  substance  acted  upon,  is  well  known.  Emil  Fischer 
investigated  this  problem  of  specificity  extensively  in  connection  with 
the  fermentation  of  sugars  and  reached  the  conclusion  that  enzymes, 
with  the  possible  exception  of  certain  oxidases,  can  act  only  upon  such 
substances  as  have  a  specific  stereo-isomeric  relationship  to  themselves. 
He  considers  that  the  enzyme  and  its  substrate  must  have  an  inter- 
relation, such  as  the  key  has  to  the  lock,  or  the  reaction  does  not  occur. 
Fischer  was  able  to  predict,  in  certain  definite  cases,  from  a  knowledge 
of  the  constitution  and  stereo-chemical  relationships  of  a  substance, 

'For  literature,  see  Kendall  and  Sherman:  Jour,  Am.  Client. Soc,  32,  10S7,  1910. 
'^  Bergeim  and  Hawk:  Unpublished  data. 
'Wohlgemuth:  Biochemische  Zeitschrift,  9,  10,  1908. 
*Bierry:  Ibid.,  40,  357,  1912. 


8  PHYISOLOGICAL    CHEMISTRY. 

whether  or  not  it  would  be  acted  upon  by  a  certain  enzyme.  An  appli- 
cation of  this  specificity  of  enzyme  action  may  be  seen  in  the  well-known 
facts  that  certain  enzymes  act  on  carbohydrates,  others  on  fats,  and  others 
on  protein;  and,  moreover,  that  the  group  of  those  which  transform  car- 
bohydrates, for  example,  is  further  subdivided  into  specific  enzymes  each 
of  which  has  the  power  of  acting  alone  upon  some  one  sugar. 

It  has  been  conclusively  shown,  in  the  case  of  certain  enzymes,^ 
at  least,  that  their  action  is  a  reversible  one  and  is,  in  all  its  main  fea- 
tures, directly  analogous  to  the  reversible  reactions  produced  by  chem- 
ical means.  For  instance,  in  the  saponification  of  ethyl-butyrate  by 
means  of  pancreatic  lipase,  it  has  been  shown  that  upon  the  formation 
of  the  end-products  of  the  reaction,  i.  e.,  butyric  acid  and  ethyl  alcohol, 
there  is  reversion^  and  the  reaction  is  stationary.     This  does  not  mean 

CjH^COO.CjHj  +  H^O^i^CgH.COOH-FCsHsOH. 

Ethyl  butyrate.  Butyric  acid.     Ethyl  alcohol. 

there  are  no  chemical  changes  going  on,  but  simply  indicates  that  chem- 
ical equilibrium  has  been  established,  and  that  the  change  in  one  direction 
is  counterbalanced  by  the  change  in  the  opposite  direction.  Pancreatic 
lipase  was  one  of  the  first  enzymes  to  have  the  reversibility  of  its  reaction 
clearly  demonstrated.^  A  knowledge  of  the  fact  that  lipase  possesses 
this  reversibility  of  action  is  of  extreme  physiological  importance  and 
aids  us  materially  in  the  explanation  of  the  processes  involved  in  the  diges- 
tion, absorption,  and  deposition  of  fats  in  the  animal  organism  (see 
p.  141). 

In  respect  to  many  enzymes  it  has  been  found  that  the  law  govern- 
ing the  action  of  inorganic  catalyzers  is  directly  applicable,  i.  e.,  that 
the  intensity  is  almost  directly  proportional  to  the  concentration  of  the  enzyme. 
In  the  case  of  enzymes,  however,  there  is  a  difference  in  that  a  maximum 
intensity  is  soon  reached  and  that  subsequent  concentration  of  the  en- 
zyme is  productive  of  no  further  increase  in  intensity.  The  enzymes  which 
have  been  shown  to  obey  this  linear  law  are  lipase,  invertase,  rennin,  and 
trypsin.  In  certain  instances,  where  this  law  of  direct  proportionality 
between  the  intensity  of  action  and  the  concentration  of  enzyme  does  not 
hold,  it  has  been  found  that  the  Schiltz-Borissow  law,  first  experimentally 
demonstrated  by  E.  Schutz,  was  applicable.  This  is  to  the  effect  that 
the  intensity  is  directly  proportional  to  the  square  root  of  the  concentra- 
tion, or  conversely,  that  the  relative  concentrations  of  enzymes  are  directly 
proportional  to  the  squares  of  the  intensities} 

'  This  is  probably  a  general  condition. 

*The  re-synthesis  of  ethyl-butyrate  from  its  hydrolysis  products.  This  may  be  indicated 
thus: 

'The  principle  was  first  demonstrated  in  connection  with  the  enzyme  maltase  (see  p.  62). 
*  This  SchUtz-Borissow  law  is  not  generally  applicable. 


ENZYMES  AND   THEIR   ACTION.  9 

It  has  been  shown  that  there  are  certain  substances  which  possess 
the  property  of  directly  inhibiting  or  preventing  the  action  of  a  catalyzer. 
These  are  called  anti-catalyzers  or  paralyzers  and  have  been  compared 
to  the  anti-toxins.  Related  to  this  class  of  anti-catalytic  agents  stand 
the  anti-enzymes.  The  first  anti-enzyme  to  be  reported  was  the  anti- 
rennin  of  Morgenroth.  This  was  produced  by  injecting  into  an  animal 
increasing  doses  of  rennet  solution,  whereupon  an  "anti"  substance 
was  subsequently  found  both  in  the  serum  and  in  the  milk,  which 
prevented  the  enzyme  rennin  from  exerting  its  normal  activity  in  the 
presence  of  caseinogen.  In  other  words,  anti-rennin  had  been  formed 
in  the  serum  of  the  animal,^  through  the  repeated  injections  of  rennet 
solution.  Since  the  discovery  of  this  anti-enzyme,  anti-bodies  have 
been  demonstrated  for  pepsin,  trypsin,  lipase,  urease,  amylase,  laccase, 
tyrosinase,  emulsin,  papain,  and  thrombin.  According  to  Weinland, 
the  reason  why  the  stomach  does  not  digest  itself  is,  that  during  life 
there  is  present  in  the  mucous  membrane  of  the  stomach  an  anti-enzyme 
{anti-pepsin)  which  has  the  property  of  inhibiting  the  action  of  pepsin. 
A  similar  substance  (anti-trypsin)  is  present  in  the  intestinal  mucosa 
as  well  as  in  the  tissues  of  various  intestinal  worms.  Some  investigators 
are  not  inclined  to  accept  the  enzyme  nature  of  these  inhibitory  agents  as 
proven. 

The  very  recent  investigations  of  Ehrlich^  and  of  Neuberg^  have 
served  to  cause  a  complete  revision  of  our  ideas  regarding  yeast  fermenta- 
tion. Ehrlich,  for  example,  has  shown  that  yeast  will  liberate  ammonia 
from  amino  acids  and  leave  behind  a  non-nitrogenous  complex.  Among 
these  complexes  amyl  alcohol,  succinic  acid  and  others  may  be  mentioned. 
Thus,  amyl  alcohol  results  from  the  fermentation  of  leucine,  whereas  ethyl 
alcohol  results  from  the  fermentation  of  sugar.  Neuberg  has  demon- 
strated the  presence  in  the  yeast  of  an  enzyme  termed  carboxylase  which  has 
the  property  of  splitting  off  carbon  dioxide  from  the  carboxyl  group  of 
amino  and  other  aliphatic  acids.  The  findings  mentioned  above  constitute 
the  basis  for  much  important  work  on  so-called  "sugar- free  fermentation." 

For  a  more  extended  consideration  of  enzymes  the  student  is  referred  to 
the  following  sources: — 

Bayliss. — The  Nature  of  Enzyme  Action,  Second  Edition,  Longmans, 
Green  and  Co.,  New  York  and  London. 

DuCLAUX. — Traite  de  Microbiologie,  Masson   &  Co.,  Paris. 

Effront. — Enzymes  and  their  Applications,  Translated  by  Prescott, 
Wiley  and  Sons,  New  York. 

'  Serum  is  normally  anti-tryptic. 
^Ehrlich:  Biochemische  Zeitschrift,  36,  477,  191 1. 

^Neuberg  and  Collaborators:  Biochemische  Zeitschrift,  31,  170;  32,  323;  36  (60,  68,  and 
76),  1911. 


lO  PHYSIOLOGICAL   CHEMISTRY. 

EuLER. — (a)  Allgemeine  Chemie  der  Enzyme,  Bergmann,  Wiesbaden, 
1910.     (b)  Ergebnisse  der  Physiologie,  1909-10. 

Oppenheimer. — Die  Fermente  und  Ihre  Wirkungen,  Dritte  Auflage, 
Vogel,  Leipzig. 

Samuely. — Handbuch  der  Biochemie  des  Menschen  und  der  Thiere 
(Oppenheimer),  Gustav  Fischer,  Jena. 

Vernon. — Intracellular  Enzymes,  Murray,  London. 

EXPERIMENTS  ON  ENZYMES  AND  ANTI-ENZYMES. 
A.  Experiments  on  Enzymes.^ 

I.  AMYLASES. 

1.  Demonstration  of  Salivary  Amylase.^ — To  25  c.c.  of  a  one 
per  cent  starch  paste  in  a  small  beaker,  add  5  drops  of  saliva  and  stir 
thoroughly.  At  intervals  of  a  minute  remove  a  drop  of  the  solution 
to  one  of  the  depressions  of  a  test-tablet  and  test  by  the  iodine  test.^ 
If  the  blue  color  with  iodine  still  forms  after  five  minutes,  add  another 
five  drops  of  saliva.  The  opalescence  of  the  starch  solution  should 
soon  disappear,  indicating  the  formation  of  soluble  starch  {amidulin) 
which  gives  a  blue  color  with  iodine.  This  body  should  soon  be  trans- 
formed into  erythro dextrin  which  gives  a  red  color  with  iodine,  and  this, 
in  turn,  should  pass  into  achroodextrin  which  gives  no  color  with  iodine. 
This  point  is  called  the  achromic  point.  When  this  point  is  reached 
test  by  Fehling's  test*  to  show  the  production  of  a  reducing  substance 
(maltose).  A  positive  Fehling's  test  may  be  obtained  whib  the  solution 
still  reacts  red  with  iodine  inasmuch  as  some  sugar  is  formed  from  the 
soluble  starch  coincidently  with  the  formation  of  the  erythrodextrin.  For 
further  discussion  of  the  transformation  of  starch  see  p.  61. 

2.  Demonstration  of  Pancreatic  Amylase/ — ^Proceed  exactly 
as  indicated  above  in  the  Demonstration  of  Salivary  Amylase  except 
that  the  saliva  is  replaced  by  5  c.c.  of  pancreatic  extract  prepared  as 
described  on  p.  153.  Pancreatic  amylase  transforms  the  starch  in  a 
manner  entirely  analogous  to  the  transformation  resulting  from  the 
action  of  salivary  amylase. 

3.  Preparation  of  Vegetable  Amylase. — Extract  finely  ground 
malt  with  water,  filter  and  subject  the  filtrate  to  alcoholic  fermentation 
by  means  of  yeast.     When  fermentation  is  complete  filter  off  the  yeast 

'  If  it  is  deemed  advisable  Ijy  the  instructor  to  give  all  the  practical  work  upon  enzymes  at 
this  point  in  the  course,  additional  experiments  will  be  found  in  Chapters  III,  VI  and  VIII. 
^  For  a  discussion  of  this  enzyme  see  p.  60. 
^See  p.  50. 
^See  p.  32. 
'  For  a  discussion  of  this  enzyme  see  p.  1 50. 


ENZYMES  AND   THEIR  ACTION.  II 

and  precipitate  the  amylase  from  the  filtrate  by  the  addition  of  alcohol. 
The  precipitate  may  be  filtered  off  and  obtained  in  the  form  of  a  fmc  white 
powder. 

4.  Demonstration  of  Vegetable  Amylase. — This  enzyme  may 
be  demonstrated  according  to  the  directions  given  under  Demonstra- 
tion of  Salivary  Amylase,  p.  10,  with  the  exception  that  the  saliva  used 
in  that  experiment  is  replaced  by  an  aqueous  solution  of  the  vegetable 
amylase  powder  prepared  as  described  above. ^ 

II.  PROTEASES. 

1.  Preparation  of  Gastric  Protease.- — Treat  the  finely  com- 
minuted mucosa  of  a  pig's  stomach  with  0.4  per  cent  hydrochloric 
acid  and  extract  at  38°  C.  for  24-48  hours.  The  filtrate  from  this  mix- 
ture constitutes  a  very  satisfactory  acid  extract  of  gastric  protease  (see 
p.  130). 

2.  Demonstration  of  Gastric  Protease. — Introduce  some  pro- 
tein material  (fibrin,  coagulated  egg-white,  or  washed  lean  beef)  into 
the  acid  extract  of  gastric  protease  prepared  as  above  described,-^  add 
an  equal  volume  of  0.4  per  cent  hydrochloric  acid  and  place  the  mix- 
ture at  38°  C.  for  2-3  days.  Identify  the  products  of  digestion  according 
to  directions  given  on  p.  130. 

3.  Preparation  of  Pancreatic  Protease.^ — A  satisfactory  ex- 
tract of  this  enzyme  may  be  made  from  the  pancreas  of  a  pig  or  sheep 
according  to  the  directions  given  on  p.  153. 

4.  Demonstration  of  Pancreatic  Protease. — Into  an  alkaline 
extract  of  pancreatic  protease,^  prepared  as  directed  on  p.  153,  introduce 
some  fibrin,  coagulated  egg-white  or  lean  beef  and  place  the  mixture 
at  38°  C.  for  2-5  days."  At  the  end  of  that  period  separate  and  identify 
the  end-products  of  the  action  of  pancreatic  protease  according  to  the 
directions  given  on  p.  153. 

5.  Demonstration  of  a  Vegetable  Protease. — A  commercial 
preparation  of  papain  {papayotin,  carase  or  papase),  the  protease  of  the 
fruit  of  the  pawpaw  {carica  papaya),  may  be  used  in  this  connection. 
Follow  the  same  procedure  as  that  described  under  Gastric  Protease 
(see  above). 

*  If  desired  the  first  aqueous  extract  of  the  original  malt  may  be  used  in  this  demonstration. 
Commercial  taka-diastase  may  also  be  employed. 

-Also  called  pepsin,  pepsase,  gastric  proteinase,  and  acid  protease.  For  a  discussion  of  this 
enzyme  see  p.  127. 

^  If  so  desired,  a  solution  of  commercial  pepsin  powder  in  0.2  per  cent,  hydrochloric  acid 
may  be  substituted. 

*  .\lso  called  trypsin,  trypsase,  pancreatic  proteinase  and  alkali  proteinase.  For  a  discussion 
of  this  enzyme  see  p.  149. 

W  0.25  per  cent  sodium  carbonate  solution  of  commercial  trypsin  may  be  substituted. 
*.\    few   c.c.    of    toluol  or  an  alcoholic  solution  of  thymol  should  be  added  to  prevent 
putrefaction. 


12  PHYSIOLOGICAL   CHEMISTRY. 

It  has  been  demonstrated  by  Mendel  and  Blood  ^  that  the  presence  of 
HCN  will  accelerate  the  proteolytic  activity  of  papain.  It  is  suggested 
that  the  HCN  acts  as  a  so-called  co-enzyme  (see  p.  7). 

.  Vines-  believes  that  "papain"  consists  of  a  mixture  of  two  enzymes, 
a  pepsin  and  an  erepsin.  Mendel  and  Blood  do  not  consider  the  evidence 
on  this  point  as  conclusive. 

III.  LIPASES. 

I.  Preparation  of  Pancreatic  Lipase.^ — An  extract  of  this  en- 
zyme may  be  prepared  from  the  pancreas  of  the  pig  or  sheep  accord- 
ing to  the  directions  given  on  p.  153.* 

.2.  Demonstration  of  Pancreatic  Lipase. — Into  each  of  two  test- 
tubes  introduce  10  c.c.  of  milk  and  a  small  amount  of  litmus  powder. 
To  the  contents  of  one  tube  add  3  c.c.  of  a  neutral  extract  of  pancreatic 
lipase  and  to  the  contents  of  the  other  tube  add  3  c.c.  of  a  boiled  neutral  ex- 
tract of  pancreatic  lipase.  Keep  the  tubes  at  38°  C.  and  watch  for  color 
changes.  The  blue  color  of  the  litmus  powder  will  gradually  give  place 
to  a  red.  This  change  in  color  of  the  litmus  from  blue  to  red  has  been 
brought  about  by  the  fatty  acid  which  has  been  produced  through  the 
lipolytic  action  exercised  by  the  lipase  upon  the  milk  fats. 

3.  Preparation  of  Vegetable  Lipase. — This  enzyme  may  be 
readily  prepared  from  castor  beans,  several  months  old,  by  the  following 
procedure:^  Grind  the  shelled  beans  very  fine®  and  extract  for  twenty- 
four-hour  periods  with  alcohol-ether  and  ether,  in  turn.  Reduce  the  semi- 
fat-free  material  to  the  finest  possible  consistency  by  means  of  mortar 
and  pestle  and  pass  this  material  through  a  sieve  of  very  fine  mesh.  Place 
this  material  in  a  Soxhlet  extractor  and  extract  for  one  week.  This 
fat-free  powder  may  then  be  used  to  demonstrate  the  action  of  vegetable 
lipase.  Powder  prepared  as  described  may  be  used  in  quantitative  tests. 
For  ordinary  qualitative  tests  it  is  not  necessary  to  remove  the  last  traces 
of  fat  and  therefore  the  extraction  period  in  the  Soxhlet  apparatus  may 
be  much  shortened. 

4.  Demonstration  of  Vegetable  Lipase. — The  lipolytic  action 
of  the  lipase  prepared  from  the  castor  bean,  as  just  described,  may 
be  demonstrated  in  a  manner  entirely  analogous  to  that  used  in  the 
Demonstration  of  Pancreatic  Lipase,  see  above.     Proceed  as  indicated 

'  Mendel  and  Blood:  Journal  of  Biological  Chemistry,  8,  177,  1910. 

'Vines:  Annals  of  Botany,  19,  174,  1905. 

'Also  called  steapsin.  For  a  discussion  of  this  enzyme  see  p.  151.  A  very  active  lipolytic 
extract  may  also  be  prepared  from  the  liver. 

*If  preferred,  a  glycerol  extract  may  be  prepared  according  to  the  directions  given  by 
Kanitz;  Zeitschriftfur  physiologische  Chemie,  1906,  46,  p.  4cS2. 

*  A.  E.  Taylor:  On  Fermetitalion;  University  of  California  Publications,  1907. 

*  The  shells  should  be  removed  without  the  use  of  water.  These  beans  are  poisonous,  due 
to  their  content  of  ricin. 


ENZYMES   AND    THEIR   ACTION,  1 3 

in  that  experiment  and  substitute  the  vegetable  lipase  powder  for  the 
neutral  extract  of  pancreatic  lipase.  The  type  of  action  is  entirely 
analogous  in  the  two  instances. 

An  experiment  similar  to  Experiment  2,  p.  157,  may  also  be  tried 
if  desired.  In  this  experiment  0.2  c.c.  of  cither  ethyl  butyrate  or  amyl 
acetate  may  be  employed. 

IV.  INVERTASES.' 

1.  Preparation  of  an  Extract  of  Sucrase.- — Treat  the  finely 
divided  epithelium  of  the  small  intestine  of  a  dog,  pig,  rat,  rabbit,  or  hen 
with  about  three  volumes  of  a  two  per  cent  solution  of  sodium  fluoride 
and  permit  the  mixture  to  stand  at  room  temperature  for  twenty-four 
hours.  Strain  the  extract  through  cloth  or  absorbent  cotton  and  use  the 
strained  material  in  the  following  demonstration. 

2.  Demonstration  of  Sucrase. — To  about  5  c.c.  of  a  one  per  cent 
solution  of  sucrose,  in  a  test-tube,  add  about  one  cubic  centimeter  of  a 
two  per  cent  sodium  fluoride  intestinal  extract,  prepared  as  described 
above.  Prepare  a  control  tube  in  which  the  intestinal  extract  is  boiled 
before  being  added  to  the  sugar  solution.  Place  the  two  tubes  at  2,^°  C. 
for  two  hours.^  Heat  the  mixture  to  boiling  to  coagulate  the  protein 
material,  filter,  and  test  the  filtrate  by  Fehling's  test  (see  p.  32).  The 
tube  containing  the  boiled  extract  should  give  no  response  to  Fehling's 
test,  whereas  the  tube  containing  the  imhoiled  extract  should  reduce  the 
Fehling's  solution.  This  reduction  is  due  to  the  formation  of  invert 
sugar  (see  p.  46)  from  the  sucrose  through  the  action  of  the  enzyme  su- 
crase which  is  present  in  the  intestinal  epithelium. 

3.  Preparation  of  Vegetable  Sucrase. — Thoroughly  grind  about 
100  grams  of  brewer's  yeast  in  a  mortar  with  sand.  Spread  the  ground 
yeast  in  thin  layers  on  glass  or  porous  plates  and  dry  it  rapidly  in  a  current 
of  dry,  warm  air.  Powder  this  dry  yeast,  extract  it  with  distilled  water 
and  filter.  Pour  the  filtrate  into  acetone,  stir  and  after  permitting  the 
acetone  mixture  to  stand  for  a  few  minutes  filter  on  a  Buchner  funnel. 
The  resulting  precipitate,  after  drying  and  pulverizing,  may  be  used  to 
demonstrate  vegetable  sucrase. 

4.  Demonstration  of  Vegetable  Sucrase. — To  about  5  c.c.  of  a  one 
per  cent  solution  of  sucrose  in  a  test-tube  add  a  small  amount  of  the 
sucrase  powder  prepared  as  directed  above.  Place  the  tube  at  2,^°  C. 
for  24-72  hours  and  at  the  end  of  that  period  test  the  solution  by  Fehling's 
test.     Reduction  indicates  that  the  active  sucrase  powder  has  transformed 

'  The  invertirig  enzymes  of  the  alimentary  tract;  Mendel  and  Mitchell:  American  Journal  of 
Physiology,  20,  81,  1907-08. 

*  For  a  discussion  of  this  enzyme  see  p.  152. 

'  If  a  positive  result  is  not  obtained  in  this  time  permit  the  digestion  to  proceed  for  a  longer 
period. 


14  PHYSIOLOGICAL    CHEMISTRY. 

the  non-reducing  sucrose  into  dextrose  and  Isevulose,  and  these  sugars, 
in  turn,  have  reduced  the  Fehling  solution. 

5.  Preparation  of  an  Extract  of  Lactase.^ — Treat  the  finely 
divided  epithelium  of  the  small  intestine  of  a  kitten,  puppy,  or  pig  embryo 
with  about  three  volumes  of  a  two  per  cent  solution  of  sodium  fluoride 
and  permit  the  mixture  to  stand  at  room  temperature  for  twenty-four 
hours.  Strain  the  extract  through  cloth  or  absorbent  cotton  and  use 
the  strained  material  in  the  following  demonstration. 

6.  Demonstration  of  Lactase.^ — To  about  5  c.c,  of  a  one  per 
cent  solution  of  lactose  in  a  test-tube  add  about  one  cubic  centimeter 
of  a  toluol-water  or  a  two  per  cent  sodium  fluoride  extract  of  the  first 
part  of  the  small  intestine^  of  a  kitten,  puppy,  or  pig  embryo  prepared  as 
described  above.  Prepare  a  control  tube  in  which  the  intestinal 
extract  is  boiled  before  being  added  to  the  sugar  solution.  Place  the  two 
tubes  at  38°  C.  for  24  hours.  At  the  end  of  this  period  add  one  cubic 
centimeter  of  the  digestion  mixture  to  5  c.c.  of  Barfoed's*  reagent  and 
place  the  tubes  in  a  boiling  water-bath.^  Examine  the  tubes  at  the  end  of 
three  minutes  against  a  black  background  in  a  good  light.  If  no  cuprous 
oxide  is  visible  replace  the  tubes  and  repeat  the  examination  at  the  end 
of  the  fo^irth  and  fifth  minutes.  If  no  reduction  is  then  observed  permit 
the  tubes  to  stand  at  room  temperature  for  5-10  minutes  and  examine 
again.® 

It  has  been  determined  that  disaccharide  solutions  will  not  reduce 
Barfoed's  reagent  until  after  they  have  been  heated  for  9-10  minutes  on  a 
boiling  water-bath  in  contact  with  the  reagent.^  Therefore  in  the  above 
test,  if  the  tube  containing  the  unboiled  extract  exhibits  any  reduction 
after  being  heated  as  indicated,  for  a  period  of  five  minutes  or  less,  and 
the  control  tube  containing  boiled  extract  shows  no  reduction,  it  may  be 
concluded  that  lactase  was  present  in  the  intestinal  extract.^ 

7.  Preparation  of  an  Extract  of  Maltase.^^ — Treat  the  finely 
divided  epithelium  of  the  small  intestine  of  a  cat,  kitten,  or  pig  {embryo  or 
adult)  with  about  three  volumes  of  a  two  per  cent  solution  of  sodium 
fluoride  and  permit  the  mixture  to  stand  at  room  temperature  for  twenty- 

1  For  a  discussion  of  this  enzyme  see  p.  152. 
^Roaf;  Bio-Chemical  Journal,  3,  182,  1908. 
'  Duodenum  and  first  part  of  jejunum. 

*  To  4.5  grams  of  neutral  crystallized  copper  acetate  in  900  c.c.  of  water,  add  0.6  c.c.  of 
glacial  acetic  acid  and  make  the  total  volume  of  the  solution  one  liter. 

*  Care  should  be  taken  to  see  that  the  water  in  the  bath  reaches  at  least  to  the  upper  level 
of  the  contents  of  the  tubes. 

"  Sometimes  the  drawing  of  conclusions  is  facilitated  by  pouring  the  mixture  from  the  tube 
and  examining  the  bottom  of  the  tube  for  adherent  cuprous  oxide. 

'The  heating  for  9-10  minutes  is  sufficient  to  transform  the  disaccharide  into  mono- 
saccharide. 

*  The  reduction  would,  of  course,  be  due  to  the  action  of  the  dextrose  and  galactose  which 
had  been  formed  from  the  lactose  through  the  action  of  the  enzyme  lactase. 

"  For  a  discussion  of  this  enzyme  see  p.  62. 


ENZYMES  AND    THEIR  ACTION.  1 5 

four  hours.     Strain  the  extract  through  cloth  and  use  the  strained  material 
in  the  followinfi;  demonstration. 

8.  Demonstration  of  Maltase. — Proceed  exactly  as  indicated  in  the 
demonstration  of  lactase,  above,  except  that  a  one  per  cent  solution  of 
maltose  is  substituted  for  the  lactose  solution.  The  extract  used  may  be 
prepared  from  the  upper  part  of  the  intestine  of  a  cat,  kitten,  or  pig  {embryo 
or  adult).  In  the  case  of  lactase,  as  indicated,  the  intestine  used  should 
be  that  of  a  kitten;* puppy,  or  pig  {embryo). 

V.  EREPSm.i 

1.  Preparation  of  Erepsin. — Grind  the  mucous  membrane  of  the 
small  intestine  of  a  cat,  dog,  or  pig  with  sand  in  a  mortar.  Treat  the 
mortared  membrane  with  toluol-  or  chloroform-water  and  permit  the 
mixture  to  stand,  with  occasional  shaking,  for  24-72  hours. ^  Filter  the 
extract  thus  prepared  through  cotton  and  use  the  filtrate  in  the  following 
experiment. 

2.  Demonstration  of  Erepsin. — To  about  5  c.c.  of  a  one  per  cent 
solution  of  Witte's  peptone  in  a  test-tube  add  about  i  c.c.  of  the  erepsin 
extract  prepared  as  described  above  and  make  the  mixture  slightly 
alkaline  (o.i  per  cent)  with  sodium  carbonate.  Prepare  a  second  tube 
containing  a  like  amount  of  peptone  solution  but  boil  the  erepsin  extract 
before  introducing  it.  Place  the  two  tubes  at  38°  C.  for  2-3  days.  At 
the  end  of  that  period  heat  the  contents  of  each  tube  to  boiling,  filter  and  try 
the  biuret  test  on  each  filtrate.  In  making  these  tests  care  should  be  taken 
to  use  like  amounts  of  filtrate,  potassium  hydroxide  and  copper  sulphate 
in  each  test  in  order  that  the  drawing  of  correct  conclusions  may  be 
facilitated.  The  contents  of  the  tube  which  contained  the  boiled  extract 
should  show  a  deep  pink  color  with  the  biuret  test,  due  to  the  peptone  still 
present.  On  the  other  hand,  the  biuret  test  upon  the  contents  of  the  tube 
containing  the  unboiled  extract  should  be  negative  or  exhibit,  at  the  most,  a 
faint  pink  or  bhie  color,  signifying  that  the  peptone,  through  the  influence 
of  the  erepsin,  has  been  transformed,  in  great  part  at  least,  into  amino 
acids  which  do  not  respond  to  the  biuret  test.^ 

3.  The  Glycyl-Tryptophane  Reaction. — The  dipeptide  glycyl- 
tryptophane*  may  be  used  in  place  of  the  peptone  solution  for  the 
demonstration  of  erepsin.  It  is  used  widely  in  the  diagnosis  of  gastric 
cancer.     It  has  been  found  that  a  peptide-splitting  enzyme  (erepsin)  is 

'  For  a  discussion  of  this  enzyme  see  p  152. 

^  The  enzyme  may  also  be  e.xtracted  by  means  of  glycerol  or  alkaline  " physiological" salt 
solution  if  desired. 

^Strictly  speaking,  this  erepsin  demonstration  is  not  adequate  unless  a  control  test  is  made 
with  native  protein  (except  caseinogen,  histones  and  protamines)  to  show  that  the  extract  is 
trypsin-free  and  digests  peptone  but  not  native  protein. 

^  This  dipeptide  is  sold  commercially  under  the  name  "Ferment  Diagnosticon." 


l6  PHYSIOLOGICAL    CHEMISTRY, 

present  in  the  stomach  contents  of  individuals  suffering  from  cancer 
of  the  stomach,  whereas  the  stomach  contents  of  normal  individuals 
contains  no  such  enzyme.  The  glycyl-tryptophane  test,  therefore,  furn- 
ishes a  means  of  aiding  in  the  diagnosis  of  this  disorder.  As  applied  to 
stomach  contents,  the  test  is  as  follows:^  Introduce  about  lo  c.c.  of  the 
filtrate  from  the  stomach  contents  into  a  test-tube,  add  a  little  glycyl- 
tryptophane,  and  a  layer  of  toluol  and  place  the  tube  in  an  incubator 
at  38°  C.  for  24  hours.  At  the  end  of  this  time  by  means  of  a  pipette 
transfer  2-3  c.c.  of  the  fluid  from  beneath  the  toluol  to  a  test-tube, 
add  a  few  drops  of  3  per  cent  acetic  acid  and  carefully  introduce  bromine 
vapors.  Shake  the  tube  and  note  the  production  of  a  red  color  if 
tryptophane  is  present.  The  tryptophane  has,  of  course,  been  liberated 
from  the  peptide  through  the  action  of  the  peptide-splitting  enzyme 
(erepsin)  elaborated  by  the  cancer  tissue. 

If  an  excess  of  bromine  is  added  the  color  will  vanish.  If  no  rose 
color  is  noted,  add  more  bromine  vapors  carefully  with  shaking  until 
further  addition  of  the  vapors  causes  the  production  of  a  yellowish  color. 
This  indicates  an  excess  of  bromine  and  constitutes  a  negative  test. 
Occasionally  the  rose  color  indicating  a  positive  test  is  so  transitory  as  to 
escape  detection  unless  the  test  be  very  carefully  performed. 

VI.  URICOLYTIC  ENZYME.2 

1.  Preparation  of  Uricase  (Uricolytic  Enz3rme). — Extract  pulped 
liver  tissue  with  toluol-  or  chloroform-water  at  2)^°  C.  for  24  hours,  with 
occasional  shaking.  Filter  the  extract  and  use  the  filtrate  in  the  following 
experiment. 

2.  Demonstration  of  Uricase  (Uricolytic  Enzyme). — Add  about 
0.1  gram  of  uric  acid  to  10  c.c.  of  water  and  bring  the  uric  acid  into  solu- 
tion by  the  addition  of  the  minimal  quantity  of  potassium  hydroxide. 
To  5  c.c.  of  this  uric  acid  solution,  in  a  test-tube,  add  5  c.c.  of  the  uricolytic 
enzyme  extract  prepared  as  described  above.  Prepare  a  second  tube 
containing  a  like  amount  of  uric  acid  solution,  but  boil  the  extract  before 
it  is  introduced.  Place  the  two  tubes  at  T)d)°  C.  for  3-4  days  and  titrate 
the  two  digestive  mixtures  with  a  solution  of  potassium  permanganate 
according  to  directions  given  under  Folin-Schaffer  Method,  Chapter 
XXII.  It  will  be  found  that  the  mixture  containing  the  boiled  extract 
requires  a  much  larger  volume  of  the  permanganate  to  complete  the 
titration  than  the  other  tube.  This  indicates  that  a  uricolytic  enzyme 
has  destroyed  at  least  a  portion  of  the  uric  acid  which  was  originally 
present  in  the  tube  containing  the  unboiled  extract. 

*  Neubauer  and  Fischer;  Deutsches  Archiv f.  klinische  Medizin,  97,  499,  1909. 

*  Mendel  and  Mitchell;  American  Journal  of  Physiology,  20,  97,  1908. 


ENZYMES  AND   THEIR  ACTION.  1 7 

VII.    CATALASE. 

Demonstration  of  Catalase. — The  various  animal  tissues,  such 
as  liver,  kidney,  blood,  lung,  muscle  and  brain,  contain  an  enzyme  called 
catalase  which  possesses  the  property  of  decomposing  hydrogen  peroxide. 
The  presence  of  this  enzyme  may  be  demonstrated  as  follows:  Introduce 
into  a  low,  broad,  wide-mouthed  bottle  some  pulped  liver  tissue  and  a 
porcelain  crucible  containing  neutral  hydrogen  peroxide.^  Connect  the 
bottle  with  a  eudiometer  filled  with  water,  upset  the  crucible  of 
hydrogen  peroxide  upon  the  liver  pulp  and  note  the  collection  of  gas 
in  the  eudiometer.  This  gas  is  oxgyen  which  has  been  liberated  from 
the  hydrogen  peroxide  through  the  action  of  the  catalase  of  the  liver 
tissue. 

See  p.  23  for  a  method  for  the  quantitative  determination  of  catalase 
based  on  the  above  principle. 

B.  Experiments  on  Anti-Enzymes. 

1.  Preparation  of  an  Extract  of  Anti-Pepsin.^ — Grind  up  a 
number  of  intestinal  worms  (ascaris)^  with  quartz  sand  in  a  mortar. 
Subject  this  mass  to  high  pressure,  filter  the  resultant  juice  and  treat 
it  with  alcohol  until  a  concentration  of  sixty  per  cent  is  reached.  If 
any  precipitate  forms  it  should  be  filtered  oflf*  and  alcohol  added  to  the 
filtrate  until  the  concentration  of  alcohol  is  85  per  cent,  or  over.  The 
anti-enzyme  is  precipitated  by  this  concentration.  Permit  this  precipitate 
to  stand  for  twenty-four  hours,  then  filter  it  off,  wash  it  with  95  per  cent 
alcohol,  absolute  alcohol,  and  ether,  in  turn,  and  finally  dry  the  substance 
over  sulphuric  acid.  The  sticky  powder  which  results  may  be  used  in 
this  form  or  may  be  dissolved  in  water  as  desired  and  the  aqueous 
solution  used.^ 

2.  Demonstration  of  Anti-Pepsin.® — Introduce  into  a  test-tube 
a  few  fibrin  shreds  and  equal  volumes  of  pepsin-hydrochloric  acid'' 
and  ascaris  extract  made  as  indicated  above.  Prepare  a  control  tube 
in  which  the  ascaris  extract  is  replaced  by  water.  Place  the  tubes  at 
^S°  C.  Ordinarily  in  one  hour  the  fibrin  in  the  control  tube  will  be  com- 
pletely digested.     The  fibrin  in  the  tube  containing  the  ascaris  extract 

*  Mendel  and  Leavenworth;  American  Journal  of  Physiology,  21,  85,  1908. 
^  Anti-gastric-protease  or  anti-acid-protease. 

'  These  may  be  readil\-  obtained  from  pigs  at  a  slaughter  house. 

*  This  precipitate  consists  of  impurities,  the  anti-enzyme  not  being  precipitated  until  a 
higher  concentration  of  alcohol  is  reached. 

*  The  original  ascaris  extract  possesses  much  greater  activity'  than  either  the  powder  or 
the  aqueous  solution. 

*  Martin  H.  Fischer;  Physiology  of  Alimentation,  1907,  p.  134. 

'  Made  by  bringing  0.015  gram  of  pepsin  into  solution  in  7  c  c.  of  water  and  o.  23  gram  of 
concentrated  hydrochloric  acid. 


16  PHYSIOLOGICAL    CHEMISTRY. 

may,  however,  remain  unchanged  for  days,  thus  indicating  the  inhibitory 
influence  exerted  by  the  anti-enzyme  present  in  this  extract. 

3.  Preparation  of  an  Extract-  of  Anti-Trypsin/ — The  extract 
may  be  prepared  from  the  intestinal  worm,  ascaris,  according  to  the 
directions  given  on  page  17. 

4.  Demonstration  of  Anti-Trypsin. — Introduce  into  a  test-tube 
a  few  shreds  of  iibrin  and  equal  volumes  of  an  artificial  tryptic  solution^ 
and  the  ascaris  extract  made  as  described  on  page  17.  Prepare  a  control 
tube  in  which  the  ascaris  extract  is  replaced  by  water.  Place  the  two 
tubes  at  38°  C.  Ordinarily  the  fibrin  in  the  control  tube  will  be  com- 
pletely digested  in  two  hours.  The  fibrin  in  the  tube  containing  the 
ascaris  extract  may,  however,  remain  unchanged  for  days,  thus  indicating 
the  inhibitory  influence  of  the  anti-enzyme. 

Blood  serum  also  contains  anti-trypsin.  This  may  be  demonstrated 
as  follows:  Introduce  equal  volumes  of  serum  and  artificial  tryptic  solu- 
tion (prepared  as  described  above)  into  a  test-tube  and  add  a  few  shreds  of 
fibrin.  Prepare  a  control  tube  containing  boiled  serum.  Place  the  two 
tubes  at  7,8°  C.  It  will  be  observed  that  the  fibrin  in  the  tube  containing 
the  boiled  serum  digests,  whereas  that  in  the  other  tube  does  not  digest. 
The  anti-trypsin  present  in  the  unboiled  serum  has  exerted  an  inhibitory 
influence  upon  the  action  of  the  trypsin. 

C.     Quantitative  Applications. 

I.  Quantitative  Determination  of  Amylolytic  Activity. — Wohl- 
gemuth's  Method.^  Arrange  a  series  of  test-tubes  with  diminishing 
quantities  of  the  enzyme  solution  under  examination,  introduce  into  each 
tube  5  c.c.  of  I  per  cent  solution  of  soluble  starch^  and  place  each  tube  at 
once  in  a  bath  of  ice-wafer.^  When  all  the  tubes  have  been  prepared  in 
this  way  and  placed  in  the  ice-water  bath  they  are  transferred  to  a  water- 
bath  or  incubator  and  kept  at  38°  C.  for  from  thirty  minutes  to  an  hour.** 

'  Anti-pancreatic-protease  or  anli-alkali-protease. 

^  Made  by  dissolving  0.04  gram  of  sodium  carbonate  and  0.015  gram  of  trypsin  in  8  c.c. 
of  water. 

^  Wohlgemuth;  Biochemische  Zeitschrift,  9,  i,  igo8. 

*  Kahlbaum's  soluble  starch  is  satisfactory.  In  preparing  the  i  per  cent,  solution,  the 
weighed  starch  powder  should  be  dissolved  in  cold  distilled  water  in  a  casserole  and  stirred 
until  a  homogeneous  suspension  is  obtained.  The  mixture  should  then  be  heated,  with  con- 
stant stirring,  until  it  is  clear.  This  ordinarily  takes  about  8-10  minutes.  .\  slightly  opaque 
solution  is  thus  obtained  which  should  be  cooled  and  made  up  to  the  proper  volume  before 
using. 

*  Ordinarily  a  series  of  six  tubes  is  satisfactory,  the  volumes  of  the  enzyme  solution  used 
ranging  from  i  c.c.  to  o.i  c.c.  and  the  measurements  being  made  by  means  of  a  i  c.c.  gradu- 
ated pipette.  Each  tube  should  be  placed  in  the  ice- water  bath  as  soon  as  the  starch  solution  is 
introduced.     It  will  be  found  convenient  to  use  a  small  wire  basket  to  hold  the  tubes. 

"  Longer  digestion  periods  may  be  used  where  it  is  deemed  advisable.  If  exceedingly 
weak  solutions  are  being  investigated,  it  may  be  most  satisfactory  to  permit  the  digestion  to 
extend  over  a  period  of  24  hours. 


ENZYMES  AND   THEIR  ACTION.  I9 

At  the  end  of  this  digestion  period  the  tubes  are  again  removed  to 
the  bath  of  ice-water  in  order  that  the  action  of  the  enzyme  may  be 
stopped. 

Dilute  the  contents  of  each  tube,  to  within  about  one-half  inch  of  the 
top,  with  water,  add  one  drop  of  a  N/io  solution  of  iodine  and  shake  the 
tube  and  contents  thoroughly.  A  series  of  colors  ranging  from  dark  blue 
through  bluish-violet  and  reddish-yellow  to  yellow,  will  be  formed.'  The 
dark  blue  color  shows  the  presence  of  unchanged  starch,  the  bluish-violet 
indicates  a  mixture  of  starch  and  erythrodextrin,  whereas  the  reddish- 
yellow  signifies  that  erythrodextrin  and  maltose  are  present  and  the 
yellow  solution  denotes  the  complete  transformation  of  starch  into  maltose. 
Examine  the  tubes  carefully  before  a  white  background  and  select  the 
last  tube  in  the  series  which  shows  the  entire  absence  of  all  blue  color,  thus 
indicating  that  the  starch  has  been  completely  transformed  into  dcxtrins 
and  sugar.  In  case  of  indecision  between  two  tubes,  add  an  extra  drop  of 
the  iodine  solution,  and  observe  them  again,  after  shaking. 

Calculation. — The  amylolytic  activity'  of  a  given  solution  is  expressed 
in  terms  of  the  acti\ity  of  i  c.c.  of  such  a  solution.  For  example,  if  it  is 
found  that  0.02  c.c.  of  an  amylolytic  solution,  acting  at  38°  C,  completely 
transformed  the  starch  in  5  c.c.  of  a  i  percent  starch  solution  in  30  minutes, 
the  amylolytic  activity  of  such  a  solution  would  be  expressed  as  follows: 

Dll'  =  2So. 

This  indicates  that  i  c.c.  of  the  solution  under  examination  possesses  the 
power  of  completely  digesting  250  c.c.  of  i  per  cent  starch  solution  in  30 
minutes  at  t,8°  C. 

Wohlgemuth  has  suggested  a  slight  alteration  in  the  above  procedure 
for  use  in  the  determination  of  the  amylase  content  of  the  feces. ^  A  mod- 
ification of  the  Wohlgemuth  procedure*  for  this  purpose  is  given  in 
the  latter  part  of  the  chapter  on  Feces. 

2.  Quantitative  Determination  of  Peptic  Activity. — (a)  Mett's 
Method. — The  determination  of  the  actual  rate  of  peptic  actix-ity  is  a  most 
important  procedure  under  certain  conditions.  Several  methods  of 
making  this  determination  are  in  use.  The  method  of  Sprigg^  is  probably 
the  most  accurate  method  yet  devised  for  this  purpose.  It  is,  however, 
too  complicated  and  time-consuming  for  clinical  purposes.  The  method 
of  Mett,  given  below,  is  very  simple  although  not  strictly  accurate.  The 
procedure  is  as  follows:  To  about  5  c.c.  of  the  gastric  juice  under  exami- 

'  See  p.  61. 

-  Designated  by  "D"  the  first  letter  of  "diastatic." 

^  Wohlgemuth;  Berliner  klinische  Wochenschrift,  47,  92,  1910. 

*  Hawk;  .Archives  of  Internal  Medicine,  8,  552,  191 1. 

*Sprigg:  Zeitschrift  fiir  physiologische  Chemie,  35,  465,  1902. 


20  PHYSIOLOGICAL   CHEMISTRY. 

nation  in  a  test-tube  add  1-3  sections  of  a  Mett  tube^  and  place  the  mixture 
at  38°  C.  for  ten  hours.  At  the  end  of  this  period,  the  tube  should  be  re- 
moved from  the  gastric  juice  and  the  length  of  the  column  of  coagulated 
albumin  which  has  been  digested  carefully  determined  by  means  of  a  low- 
power  microscope  and  a  millimeter  scale.  In  normal  human  gastric 
juice  the  upper  limit  is  4  mm.  However,  control  tests  should  always  be 
made  to  determine  the  digestibility  of  the  coagulated  albumin  in  artificial 
gastric  juice,  inasmuch  as  this  factor  will  vary  with  different  albumin 
preparations.  This  fact  of  the  variation  in  the  digestibility  has  been 
emphasized  by  the  recent  work  of  Frank.  ^  This  investigator  demon- 
strated that  the  digestibility  of  the  egg  albumin  in  the  Mett  tube  would 
vary  according  to  the  temperature  at  which  the  albumin  was  coagulated. 
Therefore  in  making  a  series  of  comparative  tests  the  albumin  in  the 
Mett  tubes  should  be  coagulated  under  uniform  conditions  in  order  to 
insure  accuracy. 

In  connection  with  this  test  the  Schiitz-Borissow  law  should  be  borne 
in  mind.  This  principle  is  to  the  effect  that  the  amount  of  proteolytic 
enzyme  present  in  a  digestion  mixture  is  proportional  to  the  square  of  the 
number  of  millimeters  of  albumin  digested.  Therefore  a  gastric  juice  which 
digests  2  mm.  of  albumin  contains /owr  times  as  much  pepsin  as  a  gastric 
juice  which  digests  only  i  mm.  of  albumin.  And  further,  if  the  quantities 
of  albumin  digested  are  2  mm.  and  3  mm.,  respectively,  the  ratio  between 
the  pepsin  values  will  be  as  4  :  9. 

It  is  claimed  by  Nirenstein  and  Schiff^  that  the  principle  of  Schiitz  does 
not  apply  to  gastric  juice  unless  this  fluid  be  diluted  with  fifteen  volumes 
of  N/20  hydrochloric  acid. 

(6)  Fuld  and  Levison's  Method. — This  test  is  founded  upon  the  fact, 
shown  by  Osborne,  that  edestin  when  brought  into  solution  in  dilute  acid 
will  change  in  its  solubility,  due  to  the  contact  with  the  acid,  and  that  a 
protean  called  edestan,  which  is  insoluble  in  neutral  fluid,  will  be  formed. 
The  procedure  is  as  follows:  Dilute  the  gastric  juice  under  examination 
with  20  volumes  of  water  and  introduce  gradually  decreasing  volumes  of  the 
diluted  juice  into  a  series*  of  narrow  test-tubes  about  i  cm.  in  diameter. 

'  In  the  preparation  of  these  tubes,  egg-white  is  diluted  with  an  equal  volume  of  water, 
the  precipitated  globulin  filtered  off  and  the  filtrate  collected  in  a  tall,  narrow  beaker  or  a 
large  test-tube.  A  bundle  of  capillary  tubes  about  lo  cm.  in  length  and  2  mm.  in  diameter 
are  now  placed  in  this  vessel  in  such  a  manner  that  they  are  completely  submerged  in  the 
albumin  solution.  After  an  examination  has  sh^nvn  that  the  tubes  are  completely  filled  with 
the  albumin  solution  and  that  there  are  no  interfering  air-bubbles,  the  vessel  and  its  contained 
tubes  is  heated  for  5-15  minutes  in  a  boiling  water-bath,  in  order  to  coagulate  the  albumin. 
When  this  coagulation  is  complete,  the  tubes  are  removed,  all  albumin  adhering  to  them  is 
carefully  cleaned  off,  and  the  tubes  rendered  air-tight  by  the  application  of  sealing  wax  at 
either  end.     When  needed  for  use,  these  tubes  are  cut  into  sections  about  2  cm.  in  length. 

^  Frank:  Journal  0/  Biological  Chemistry,  g,  463,  191 1. 

*  Nirenstein  and  Schiff;  Archiv.  fur  Verdauungekrankheiten,  8,  559,  1902. 

*  The  longer  the  series,  the  more  accurate  the  deductions  which  may  be  drawn. 


ENZYMES   AND    THEIR  ACTION.  21 

The  measurements  of  gastric  juice  may  conveniently  be  made  with  a  i 
c.c.  pipette  which  is  accurately  graduated  in  i/ioo  c.c.  Into  the  first 
tube  in  the  scries  may  be  introduced  i  c.c.  of  gastric  juice,  and  the  tubes 
which  follow  in  the  scries  may  receive  volumes  which  differ,  in  each 
instance,  from  the  volume  introduced  into  the  preceding  tube  by  i/ioo, 
1/50,  1/20,  or  i/io  of  a  cubic  centimeter.  Now  rapidly  introduce  into 
each  tube  the  same  volume  {e.  g.,  2  c.c.)  of  a  i  :  1000  solution  of  edestin^ 
and  place  the  tGbes  at  40°  C.  for  one-half  hour.  At  the  end  of  this  time 
stratify  ammonium  hydroxide  upon  the  contents  of  each  tube,^  place 
the  tubes  in  position  before  a  black  background  and  examine  them 
carefully.  The  ammonium  hydroxide,  by  diffusing  into  the  acid  fluid, 
forms  a  neutral  zone  and  in  this  zone  will  be  precipitated  any  undigested 
edestan  which  is  present.  Select  the  tube  in  the  series  which  contains  the 
least  amount  of  gastric  juice  and  which  exhibits  no  ring,  signifying  that  the 
the  edestan  has  been  completely  digested,  and  calculate  the  peptic  activity 
of  the  gastric  juice  under  examination  on  the  basis  of  the  volume  of 
gastric  juice  used  in  this  particular  tube. 

Ca/cw/a//(W.^Multiply  the  number  of  c.c.  of  cdestin  solution  used  by 
the  dilution  to  which  the  gastric  juice  was  originally  subjected  and  divide 
the  volume  of  gastric  juice  necessary  to  completely  digest  the  edestan  by 
this  product.  For  example,  if  2  c.c.  of  the  edestin  solution  was  com- 
pletely digested  by  0.25  c.c.  of  a  1:20  gastric  juice  we  would  have  the 
following  expression:  0.25^20X2  or  1:160.  This  peptic  activity  may 
be  expressed  in  several  ways,  e.  g.,  (a)  1:160  pepsin;  (b)  160  pepsin  con- 
tent; (c)  160  parts. 

(c)  Rose's  Modification^  of  the  Jacoby-Solms  Method/— Dissolve 
0.25  gram  of  the  globulin  of  the  ordinary  garden  pea,®  Pisum  sativum, in 

*  This  edestin  should  be  prepared  in  the  usual  way  (see  p.  109),  and  brought  into  solution 
in  a  dilute  hydrochloric  acid  of  approximately  the  same  strength  as  that  which  occurs  normally 
in  the  human  stomach.  This  may  be  conveniently  made  by  adding  30  c.c.  of  N/io  hydro- 
chloric acid  to  70  c.c.  of  water.  Ordinarily  it  should  not  take  longer  than  one  minute  to 
introduce  the  edestin  solution  into  the  entire  series  of  tubes.  However,  if  the  edestin  is  added 
to  the  tubes  in  the  same  order  as  the  ammonium  hydroxide  is  afterward  stratified,  no  appreci- 
able error  is  introduced 

^  Making  the  stratification  in  the  same  order  as  the  edestin  solution  was  added. 
^  Rose:  Archives  of  Internal  Medicine,  5,  459,  1910. 

*  Sohns:  Zeitschrift  fiir  klinische  Medizin,  64,  159,  1907. 

*  The  globulin  may  be  prepared  as  follows:  "The  finely  ground  peas,  freed  as  much  as 
possible  from  the  outer  coating,  are  repeatedly  extracted  with  large  quantities  of  10  per  cent 
sodium  chloride  solution,  the  extracts  combined,  strained  through  fine  bolting-cloth,  and 
allowed  to  stand  over  night  in  large  cylinders  to  deposit  insoluble  matter.  The  supernatant 
fluid  is  siphoned  off  and  saturated  with  ammonium  sulphate.  The  precipitate  of  albumin  and 
globulin  is  filtered  off,  suspended  in  a  litUe  water,  and  dialyzed  in  running  water  for  three  days, 
until  the  salt  has  been  removed,  and  the  albumins  have  been  dissolved.  The  globulins  are 
filtered  off  and  washed  two  or  three  times  to  remove  the  last  trace  of  albumins.  To  purify 
further,  the  precipitate  is  extracted  with  10  per  cent  sodium  chloride  solution,  and  filtered 
until  perfectly  clear.  The  resulting  solution  is  neutralized  to  litmus  paper  by  the  cautious 
addition  of  dilute  sodium  hydroxide,  and  again  dialyzed  in  running  water  for  three  days  to 
remove  the  salts  completely.  The  precipitated  globulins  are  then  filtered  off  and  dried  on  a 
water-bath  at  40°  C.  During  the  entire  process  of  separation  the  proteins  should  be  preserved 
with  a  mixture  of  alcoholic  thymol  and  toluol."  This  dried  globulin  is  used  in  the  clinical 
procedure. 


22  PHYSIOLOGICAL    CHEMISTRY. 

loo  c.c.  of  lo  per  cent  sodium  chloride  solution,  warming  slightly  if 
necessary/  Filter  and  introduce  i  c.c.  of  the  clear  filtrate  into  each  of  a 
scries  of  six^  test-tubes  about  i  cm.  in  diameter.  Introduce  into  each 
tube  I  c.c.  of  0.6  per  cent  hydrochloric  acid  and  permit  a  period  of  about 
five  minutes  to  elapse  for  the  development  of  the  turbidity.  Make  a 
known  volume  of  the  gastric  juice  (5-10  c.c.  is  sufl&cient)  exactly  neutral 
to  litmus  paper  with  dilute  alkali;  and  record  the  volume  of  the  alkali  so 
used.  If  acid  metaprotein  precipitates,  filter  it  off;  if  there  is  no  precipi- 
tate proceed  without  filtration.  Dilute  the  clear  neutral  solution  with  a 
known  quantity  of  distilled  water  (usually  five  volumes)  making  proper 
allowance  for  the  volume  of  alkali  used  in  the  neutralization.  Boil  5-10 
c.c.  of  the  diluted  juice,  filter  and  add  the  following  decreasing  volumes 
(c.c.)  to  the  series  of  six  tubes:  i.o,  0.9,  0.7,  0.5,  0.2,  0.0.  Make  the 
measurements  by  means  of  a  i  c.c.  pipette  graduated  in  o.oi  c.c.  Now 
rapidly  introduce  the  imboiled,  diluted  juice  in  the  following  increasing 
volumes  (c.c.)  in  order:  0.0,  o.i,  0.3,  0.5, 0.8,  i.o.  Each  tube  now  contains 
a  total  volume  of  3  c.c.  and  a  total  acidity  of  0.2  per  cent  hydrochloric 
acid.  Shake  each  tube  thoroughly  and  place  them  at  50-52°  C.  for  fifteen 
minutes  or  at  35-36°  C.  for  one  hour.  Examine  the  series  of  tubes  at  the 
end  of  the  digestion  period  and  select  that  tube  which  contains  the  smallest 
quantity  of  gastric  juice  and  which  shows  no  turbidity.  The  volume  of 
the  juice  used  in  this  tube  is  taken  as  the  basis  for  the  calculation  of  the 
peptic  activity. 

Calculation. — The  peptic  activity  is  expressed  in  terms  of  i  c.c.  of  the 
undiluted  juice.  For  example,  if  it  requires  0.5  c.c.  of  the  diluted  juice 
(five-fold  dilution)  to  clear  up  the  turbidity  in  i  c.c.  of  the  globulin  solu- 
tion in  the  proper  experimental  time  interval  (15  minutes  or  one  hour 
according  to  temperature)  the  peptic  activity  would  be  expressed  as 
follows : 

(I-^o.5)X5  =  Io  (peptic  activity) . 

According  to  this  scale  of  pepsin  units  10  may  be  considered  as 
"normal"  peptic  activity.  These  units  are  about  i/io  as  large  as  those 
expressed  by  the  Jacoby-Solms  scale. 

Inasmuch  as  it  has  been  shown^  that  blood  serum  contains  an  anti- 
pepsin  it  is  advisable  to  test  the  gastric  juice  for  blood  before  determining 
its  proteolytic  power. 

3.  Quantitative  Determination  of  Tryptic  Activity. — Gross' 
Method. — This  method  is  based  upon  the  principle  that  faintly  alkaline 

'  This  solution  may  be  preserved  at  least  two  months  under  toluol. 

^  A  longer  series  of  tubes  may  be  used  if  desired.     However,  experience  has  shown  that  a 
series  of  six  ordinarily  affords  sufTicient  range  for  all  diagnostic  purposes. 
^  Oguro:  Biochemische  Zeitschrifl,  22,  266,  1909. 


ENZYMES   AND    THEIR   ACTION.  23 

solutions  of  casein  are  precipitated  upon  the  addition  of  dilute  (i  per  cent) 
acetic  acid  whereas  its  digestion  products  are  not  so  precipitated.  The 
method  follows:  Prepare  a  series  of  tubes  each  containing  lo  c.c.  of  a 
0.1  per  cent  solution  of  pure,  fat-free  casein/  which  has  been  heated  to  a 
temperature  of  40°  C.  Add  to  the  contents  of  the  series  of  tubes  increas- 
ing amounts  of  the  trypsin  solution  under  examination,^  and  place  them 
at  40°  C.  iov  fifteen  minutes.  At  the  end  of  this  time  remove  the  tubes  and 
acidify  the  conteftts  of  each  with  a  few  drops  of  dilute  (i  per  cent)  acetic 
acid.  The  tubes  in  which  the  casein  is  completely  digested  will  remain 
clear  when  acidified,  while  those  tubes  which  contain  undigested  casein 
will  become  more  or  less  turbid  under  these  conditions.  Select  the  first 
tube  in  the  series  which  exhibits  no  turbidity  upon  acidification,  thus 
indicating  complete  digestion  of  the  casein,  and  calculate  the  tryptic 
acti\ity  of  the  enzyme  solution  under  examination. 

Calculatian. — The  unit  of  tryptic  activity  is  an  expression  of  the  power 
of  I  c.c.  of  the  fluid  under  examination  exerted  for  a  period  of  fifteen 
minutes  on  10  c.c.  of  a  o.i  per  cent  casein  solution.  For  example,  if  0.5 
c.c.  of  a  trypsin  solution  completely  digests  10  c.c.  of  a  o.i  per  cent  solu- 
tion of  casein  in  fifteen  minutes  the  acti\'ity  of  that  solution  would  be 
expressed  as  follows: 

Tryptic  acti\'ity==  i-=-o.5  =  2. 

Such  a  trypsin  solution  would  be  said  to  possess  an  activity  of  2.  If 
0.3  c.c.  of  the  trypsin  solution  had  been  required  the  solution  would  be 
said  to  possess  an  activity  of  2,-y,  i-  ^•,  i  -=-o-3  ==3-3- 

4.  Quantitative  Determination  of  Catalase.^^In  the  determination 
of  the  catalase  values  of  tissues  weighed  portions  of  the  tissue  under 
examination  should  be  ground  with  sand  in  a  mortar  then  treated  with 
four  volumes  of  chloroform  water  and  permitted  to  extract  for  24  hours  at 
room  temperature.  An  apparatus  such  as  that  shown  in  Fig.  i  may  be 
employed  in  determining  the  catalase  values.  The  main  features  of  the 
apparatus  are  based  upon  those  of  a  delivery  funnel  for  introducing  liquids 
under  increased  or  diminished  pressure. 

In  making  a  determination  introduce  a  measured  volume  (1-4  c.c.) 
of  the  filtered  extract*  into  the  small  fliask  and  insert  the  modified  Johnson 
burette  graduated  to  5  c.c.  and  containing  50  c.c.  of  hydrogen  peroxide 
(Oakland  dioxygen  neutral^  to  congo  red)  into  the  neck  of  the  flask. 

*  Made  by  dissolving  one  gram  of  Griibler's  casein  in  a  liter  of  o.i  per  cent  sodium  car- 
bonate.    A  little  chloroform  may  be  added  to  prevent  bacterial  action. 

^  The  amount  of  solution  used  may  var}'  from  o.i-i  c.c.  The  measurements  may  con- 
veniently be  made  by  means  of  a  i  c.c.  graduated  pipette. 

'Hawk:  Journal  of  American  Chemical  Society,  ^;j,  425,  191 1. 

*  If  less  than  4  c.c.  of  extract  are  used  the  volume  should  be  made  up  to  4  c.c.  by  the  addi- 
tion of  distilled  water. 

*  An  acid  reaction  modifies  the  rate  of  the  oxygen  evolution.  (See  Mendel  and  Leaven- 
worth, American  Journal  of  Physiology,  21,  85,  1908.) 


24 


PHYSIOLOGICAL   CHEMISTRY, 


Shake  the  contents  of  the  flask  briskly^  and  record  the  volume  of  oxygen 
evolved  in  a  two-minute  period  taking  readings  at  intervals  of  fifteen 
seconds. 

Calculation. — When  a  series  of  comparative  tests  are  made  on  different 
tissues  or  on  the  same  tissue  under  different  conditions  it  is  considered 
satisfactory  to  make  a  comparison  of  the  catalase  values  upon  the  basis 


Fig.  I. — ^Apparatus  for  Quantitative  Determination  of  Catalase. 

of  the  volume  of  oxygen  evolved  in  a  period  of  two  minutes  from  5  c.c.  of 
neutral  hydrogen  peroxide  by  means  of  i  c.c.  of  a  1:4  chloroform-water 
extract  of  the  tissue. 

'  In  making  a  series  of  comparative  tests  it  is  essential  that  tlie  shaking  process  should  be 
uniform  from  determination  to  determination. 


CHAPTER  II. 
CARBOHYDRATES. 

The  name  carbohydrates  is  given  to  a  class  of  bodies  which  are  an 
especially  prominent  constituent  of  plants  and  which  are  found  also  in  the 
animal  body  either  free  or  as  an  integral  part  of  various  proteins.  They 
are  called  carbohydrates  because  they  contain  the  elements  C,  H  and  O; 
the  H  and  O  being  present  in  the  proportion  to  form  water.  The  term 
is  not  strictly  appropriate  inasmuch  as  there  are  bodies,  such  as  acetic 
acid,  lactic  acid  and  inosite,  which  have  H  and  O  present  in  the  proportion 
to  form  water,  but  which  are  not  carbohydrates,  and  there  are  also  true 
carbohydrates  which  do  not  have  H  and  O  present  in  this  proportion,  e.  g., 
rhamnose,  C^H^fi^. 

Chemically  considered,  the  carbohydrates  are  aldehyde  or  ketone 
derivatives  of  complex  alcohols.  Treated  from  this  standpoint,  the 
aldehyde  derivatives  are  spoken  of  as  aldoses,  and  the  ketone  derivatives 
are  spoken  of  as  ketoses.  The  carbohydrates  are  also  frequently  named 
according  to  the  number  of  oxygen  atoms  present  in  the  molecule,  e.  g., 
trioses,  pentoses,  and  hexoses. 

The   more   common   carbohydrates   may   be   classified   as    follows: 

I.  Monosaccharides. 

1.  Hexoses,  CgHjjOg. 

(a)  Dextrose. 

(b)  Laevulose. 

(c)  Galactose. 

2.  Pentoses,  CgHj^O.. 

(a)  Arabinose. 

(b)  Xylose. 

(c)  Rhamnose  (Methyl-pentose),  CgHj^Og. 

II.  Disaccharides,  C^^li^^O^^. 

1.  Maltose. 

2.  Lactose. 

3.  Iso-Maltose. 

4.  Sucrose. 

III.  Trisaccharides,  C^^H^fi^^. 

I.  Raffinose. 

25 


26  PHYSIOLOGICAL   CHEMISTRY. 

IV.  Polysaccharides,  (CeH^gOg)^. 

1.  Starch  Group. 

(a)  Starch. 

(b)  Inulin. 

(c)  Glycogen. 

(d)  Lichenin. 

2.  Gum  and  Vegetable  Mucilage  Group. 

(a)  Dextrin. 

(b)  Vegetable  Gums. 

3.  Cellulose  Group. 

(a)  Cellulose. 

(b)  Hemicelluloses. 

(i)  Pentosans. 

Gum  Arabic. 
(2)  Hexosans. 

Galactans. 
Agar-agar. 

Each  member  of  the  above  carbohydrate  classes,  except  the  members 
of  the  pentose  group,  may  be  supposed  to  contain  the  group  CgH^^Og, 
called  the  saccharide  group.  The  polysaccharides  consist  of  this  group 
alone  taken  a  large  number  of  times,  whereas  the  disaccharides  may  be 
supposed  to  contain  two  such  groups  plus  a  molecule  of  water,  and  the 
monosaccharides  to  contain  one  such  group  plus  a  molecule  of  water. 
Thus,  (CgHjQ05)^  =  polysaccharide,  (CqHjo05)2  +  H20— 'disaccharide, 
CgHj^Og  +  HgO-^monosaccharide.  In  a  general  way  the  solubility  of 
the  carbohydrates  varies  with  the  number  of  saccharide  groups  present, 
the  substances  containing  the  largest  number  of  these  groups  being  the 
least  soluble.  This  means  simply  that,  as  a  class,  the  monosaccharides 
(hexoses)  are  the  most  soluble  and  the  polysaccharides  (starches  and 
cellulose)  are  the  least  soluble. 

MONOSACCHARIDES. 

Hexoses,  CgH^^Og. 

The  hexoses  are  monosaccharides  containing  six  oxygen  atoms  in  a 
molecule.  They  are  the  most  important  of  the  simple  sugars,  and  two  of 
the  principal  hexoses,  dextrose  and  Isevulose,  occur  widely  distributed  in 
plants  and  fruits.  Of  these  two  hexoses,  dextrose  results  from  the 
hydrolysis  of  starch  whereas  both  dextrose  and  laevulose  are  formed  in 
the  hydrolysis  of  sucrose.  Galactose,  which  with  dextrose  results  from 
the  hydrolysis  of  lactose,  is  also  an  important  hexose.     These  three  hexoses 


CARBOHYDRATES.  27 

are  fermentable  by  yeast,  and  yield  laevulinic  acid  upon  heating  with 
dilute  mineral  acids.  They  reduce  metallic  oxides  in  alkaline  solution, 
are  optically  active  and  extremely  soluble.  With  phenylhydrazine  they 
form  characteristic  osazones. 

CH3OH 

i 
DEXTROSE,    (CHOH),. 

I 

CHO 

Dextrose,  also  called  glucose  or  grape  sugar,  is  present  in  the  blood 
in  small  amount  and  may  also  occur  in  traces  in  normal  urine.  After 
the  ingestion  of  large  amounts  of  sucrose,  lactose  or  dextrose,  causing  the 
assimilation  limit  to  be  exceeded,  an  alimentary  glycosuria  may  arise. 
The  assimilation  limit  for  dextrose  has  been  shown ^  to  be  between  loo 
and  150  grams.  In  diabetes  mellitus  very  large  amounts  of  dextrose  arc 
excreted  in  the  urine.  The  following  structural  formula  has  been 
suggested  by  Victor  Meyer  for  (f-dextrose: 

COH 

I 
H— C— OH 

I 
HO— C— H 

H— C— OH 

H— C— OH 

I 
CH.OH 

(For  further  discussion  of  dextrose  see  section  on  Hexoses,  page  26.) 

Experiments  on  Dextrose. 

1.  Solubility. — Test  the  solubility  of  dextrose  in  the  "ordinary 
solvents"  and  in  alcohol.  (In  the  solubility  tests  throughout  the  book  we 
shall  designate  the  following  solvents  as  the  "ordinary  solvents":  H^O; 
10  per  cent  NaCl;  0.5  per  cent  NajCOg;  0.2  per  cent  HCl;  concentrated 
KOH;  concentrated  HCl.) 

2.  Molisch's  Reaction. — Place  approximately  5  c.c.  of  concentrated 
HjSO^  in  a  test-tube.  Incline  the  tube  and  slowly  pour  down  the  inner 
side  of  it  approximately  5  c.c.  of  the  sugar  solution  to  which  2  drops  of 
Molisch's  reagent  (a  15  per  cent  alcoholic  solution  of  a-naphthol)  has  been 
added,  so  that  the  sugar  solution  will  not  mix  with  the  acid.     A  reddish- 

'  Brasch:  Zeitschrift  fur  Biologic,  50,  113,  1907. 


28  PHYSIOLOGICAL   CHEMISTRY. 

violet  zone  is  produced  at  the  point  of  contact.  The  reaction  is  due  to  the 
formation  of  furfurol, 

HC— CH 

HC     C-CHO, 

•       \/ 
O 

by  the  acid.  The  test  is  given  by  all  bodies  containing  a  carbohydrate 
group  and  is  therefore  not  specific  and,  in  consequence,  of  very  little 
practical  importance. 

3,  Phenylhydrazine  Reaction. — Test  according  to  one  of  the  follow- 
ing methods:  (a)  To  a  small  amount  of  phenylhydrazine  mixture, 
furnished  by  the  instructor,^  add  5  c.c.  of  the  sugar  solution,  shake  well 
and  heat  on  a  boiling  water-bath  for  one-half  to  three-quarters  of  an  hour. 
Allow  the  tube  to  cool  slowly  and  examine  the  crystals  microscopically 
(Plate  III,  opposite).  If  the  solution  has  become  too  concentrated  in  the 
boiling  process  it  will  be  light  red  in  color  and  no  crystals  will  separate 
until  it  is  diluted  with  water. 

Yellow  crystalline  bodies  called  osazones  are  formed  from  certain 
sugars  under  these  conditions,  in  general  each  individual  sugar  giving 
rise  to  an  osazone  of  a  definite  crystalline  form  which  is  typical  for  that 
sugar.  It  is  important  to  remember  in  this  connection  that  of  the  simple 
sugars  of  interest  in  physiological  chemistry,  dextrose  and  laevulose  yield 
the  same  osazone.  Each  osazone  has  a  definite  melting-point  and  as  a 
further  and  more  accurate  means  of  identification  it  may  be  recrystallized 
and  identified  by  the  determination  of  its  melting-point  and  nitrogen 
content.  The  reaction  taking  place  in  the  formation  of  phenyldextrosazone 
is  as  follows: 

Dextrose.  Phenylhydrazine.  Phenyldextrosazone. 

(6)  Place  5  c.c.  of  the  sugar  solution  in  a  test-tube,  add  i  c.c.  of  the 
phenylhydrazine-acetate  solution  furnished  by  the  instructor,^  and  heat 
on  a  boiling  water-bath  for  one-half  to  three-quarters  of  an  hour.  Allow 
the  liquid  to  cool  slowly  and  examine  the  crystals  microscopically  (Plate 
III,  opposite). 

The  phenylhydrazine  test  has  been  so  modified  by  Cipollina  as  to  be  of 
use  as  a  rapid  clinical  lest.  The  directions  for  this  test  are  given  in  the 
next  experiment. 

*  This  mixture  is  prepared  by  combining  one  part  of  phenylhydrazine  hydrochloride  and 
two  parts  of  sodium  acetate,  by  weight.     These  are  thoroughly  mixed  in  a  mortar. 

^  This  solution  is  prepared  by  mixing  one  part  by  volume,  in  each  case,  of  glacial  acetic 
acid,  one  part  of  water  and  two  parts  of  phenylhydrazine  (the  base). 


PLATF.   III. 


OSAZONES. 

Upper  form,  de.xtrosazone;  tentral  form,  maltosazonc;  lower  form,  lactosazone. 


CARBOHYDRATES.  29 

4.  CipoUina's  Test. — Thoroughly  mix  4  c.c.  of  dextrose  solu- 
tion, 5  drops  of  phenylhydrazine  (the  base)  and  1/2  c.c.  of  glacial  acetic 
acid  in  a  test-tube.  Heat  the  mixture  for  about  one  minute  over  a  low 
flame,  shaking  the  tube  continually  to  prevent  loss  of  fluid  by  bumping. 
Add  4-5  drops  of  sodium  hydroxide  (sp.  gr.  1.16),  being  certain  that 
the  fluid  in  the  test-tube  remains  acid,  heat  the  mixture  again  for  a  moment 
and  then  cool  the  contents  of  the  tube.  Ordinarily  the  crystals  form 
at  once,  especially  if  the  sugar  solution  possesses  a  low  specific  gravity. 
If  they  do  not  appear  immediately  allow  the  tube  to  stand  at  least  20 
minutes  before  deciding  upon  the  absence  of  sugar. 

Examine  the  crystals  under  the  microscope  and  compare  them  with 
those  shown  in  Plate  III,  opposite  page  28. 

5.  Riegler's  Reaction.^ — Introduce  o.i  gram  of  phenylhydra- 
zine-hydrochloride  and  0.25  gram  of  sodium  acetate  into  a  test-tube, 
add  20  drops  of  the  solution  under  examination  and  heat  the  mixture 
to  boiling.  Now  introduce  10  c.c.  of  a  3  per  cent  solution  of  potassium 
hydroxide  and  gently  shake  the  tube  and  contents.  If  the  solution 
under  examination  contains  dextrose  the  liquid  in  the  tube  will  assume 
a  red  color.  One  per  cent  dextrose  yields  an  immediate  color  whereas 
0.05  per  cent  yields  the  color  only  after  the  lapse  of  a  period  of  one- 
half  hour  from  the  time  the  alkali  is  added.  In  urinary  examination 
if  the  color  appears  after  the  thirty-minute  interval  the  color  change  is 
without  significance  inasmuch  as  sugar-free  urine  will  respond  thus. 
The  reaction  is  given  by  all  aldehydes  and  therefore  the  test  cannot 
be  safely  employed  in  testing  urines  preserved  by  formaldehyde.  Al- 
bumin does  not  interfere  with  the   test. 

6.  Bottu's  Test.^ — To  8  c.c.  of  Bottu's  reagent'  in  a  test-tube  add 
I  c.c.  of  the  solution  under  examination  and  mix  the  liquids  by  gentle 
shaking.  Now  heat  the  upper  portion  of  the  mixture  to  boiling,  add 
an  additional  i  c.c.  of  the  solution  and  heat  the  mixture  again  imme- 
diately. The  appearance  of  a  blue  color  accompanied  by  the  precipi- 
tation of  small  particles  of  indigo  blue  indicates  the  present  of  dextrose 
in  the  solution  under  examination.  The  test  will  serve  to  detect  the 
presence  of  o.i  per   cent  of  dextrose. 

7.  Precipitation  by  Alcohol. — To  10  c.c.  of  95  per  cent  alcohol 
add  about  2  c.c.  of  dextrose  solution.  Compare  the  result  with  that 
obtained  under  Dextrin,  7,  page  53. 

8.  Iodine  Test. — Make  the  regular  iodine  test  as  given  under  Starch, 


'Riegler;  Compt.  rend.  soc.  biol.,  66,  p.  795. 
-Bottu;  Compt.  rend.  soc.  bid.,  66,  p.  972. 

'This  reagent  contains  3.5  grams  of  o-nitrophenylpropiolic  acid  and  5  c.c.  of  a  freshly 
prepared  10  per  cent  solution  of  sodium  hydro.xide  per  liter. 


30  PHYSIOLOGICAL    CHEMISTRY. 

5,  page  50,  and  keep  this  result  in  mind  for  comparison  with  the  results 
obtained  later  with  starch  and  with  dextrin. 

9.  Diffusibility  of  Dextrose. — Test  the  diffusibility  of  dextrose 
solution  through  animal  membrane,  or  parchment  paper,  making  a 
dialyzer  like  one  of  the  models  shown  in  Fig.  2. 

A  most  satisfactory  dialyzing  bag  may  be  made  of  collodion  as  follows: 
Pour  a  solution  of  collodion  into  a  clean  dry  Erlenmeyer  flask  or  test- 
tube.  While  rotating  the  vessel  on  its  longitudinal  axis,  gradually  pour 
out  the  collodion,  at  the  same  time  being  careful  that  the  interior  surface 
of  the  flask  is  completely  coated  with  the  solution.  Continue  the  rotation 
in  the  inverted  position  until  the  collodion  ceases  to  flow.  After  the 
solution  has  evaporated  such  that  the  collodion  skin  on  the  rim  is  dry 


Fig.  2. — -Dialyzing  Apparatus  for  Students'  Use. 


and  stiff,  cut  or  loosen  it  around  the  edge  of  the  rim.  With  a  pipette 
or  wash  bottle  run  in  a  few  cubic  centimeters  of  water  between  the  mem- 
brane and  the  wall  of  the  flask  or  test-tube.  Shake  the  inclined  vessel 
while  rotating  on  its  longitudinal  axis,  thus  detaching  the  membrane. 
Now  withdraw  the  detached  bag  and  fill  with  water,  to  determine  whether 
or  not  it  contains  defects.^ 

TO.  Moore's  Test. — To  2-3  c.c.  of  sugar  solution  in  a  test-tube 
add  an  equal  volume  of  concentrated  KOH  or  NaOH,  and  boil.  The 
solution  darkens  and  finally  assumes  a  brown  color.  At  this  point  the 
odor  of  caramel  may  be  detected.  This  is  an  exceedingly  crude  test 
and  is  of  little  practical  value.  The  brown  color  is  due  to  the  oxida- 
tion of  the  dextrose  and  the  resulting  formation  of  the  potassium  or 
sodium  salts  of  certain  organic  acids  which  are  formed  as  oxidation 
products. 

II.  Reduction  Tests.— To  their  aldehyde  or  ketone  structure 
many  sugars  owe  the  property  of  readily  reducing  alkaline  solutions 
of  the  oxides  of  metals  like  copper,  bismuth  and  mercury;  they  also 

'  Gies:  Quoted  by  Clark.     Bioch.     Bull.,     i,   198,   1911 


CARBOHYDRATES.  3 1 

possess  the  property  of  reducing  ammoniacal  silver  solutions  with  the 
separation  of  metallic  silver.  Upon  this  property  of  reduction  the 
most  widely  used  tests  for  sugars  are  based.  When  whitish-blue  cu- 
pric  hydroxide  in  suspension  in  an  alkaline  liquid  is  heated  it  is  con- 
verted into  insoluble  black  cupric  oxide,  but  if  a  reducing  agent  like 
certain  sugars  be  present  the  cupric  hydroxide  is  reduced  to  insoluble 
yellow  cuprous  hydroxide,  which  in  turn,  on  further  heating,  may  be 
converted  into  blownish-red  or  red  cuprous  oxide.  These  changes 
are  indicated  as  follows: 

OH 

/ 
Cu  -^Cu^O  +  H^O. 

\  Cupric  oxide 

\  (black). 

OH 

Cupric  hydroxide 
(whitish- blue). 

OH 

/ 
Cu 

\ 
OH 


—  2Cu-0H  +  H,0  +  0. 
OH 


Cuprous  hydroxide 
(yellow>. 


Cu 

\ 
OH 

Cu— OH 
Cu— OH 


Cu 

\ 
O  +  H.,0. 

/ 
Cu 


Cuprous  hydro.vide  C'uprou.s  oxide 

(yellow).  (brownish-red). 

The  chemical  equations  here  discussed  are  exemplified  in  Trom- 
mer's  and  Fehling's  tests. 

(a)  Trommer's  Test. — To  5  c.c.  of  sugar  solution  in  a  test-tube 
add  one-half  its  volume  of  KOH  or  NaOH.  Mix  thoroughly  and 
add,  drop  by  drop,  a  very  dilute  solution  of  copper  sulphate.  Con- 
tinue the  addition  until  there  is  a  slight  permanent  precipitate  of  cupric 
hydroxide  and  in  consequence  the  solution  is  slightly  turbid.  Heat, 
and  the  cupric  hydroxide  is  reduced  to  yellow  cuprous  hydroxide  or 
to  brownish-red  cuprous  oxide.  If  the  solution  of  copper  sulphate  used 
is  too  strong  a  small  brownish-red  precipitate  produced  in  a  weak  sugar 


32  PBT^SIOLOGICAL   CHEMISTRY. 

solution  may  be  entirely  masked.  On  the  other  hand,  particularly 
in  testing  for  sugar  in  the  urine,  if  too  little  copper  sulphate  is  used  a 
light-colored  precipitate  formed  by  uric  acid  and  purine  bases  may 
obscure  the  brownish-red  precipitate  of  cuprous  oxide.  The  action  of 
KOH  or  NaOH  in  the  presence  of  an  excess  of  sugar  and  insufficient 
copper  will  produce  a  brownish  color.  Phosphates  of  the  alkaline 
earths  may  also  be  precipitated  in  the  alkaline  solution  and  be  mistaken 
for  cuprous  hydroxide.     Trommer's  test  is  not  very  satisfactory. 

Salkowski  ^  has  very  recently  proposed  a  modification  of  the  Trommer 
procedure  which  he  claims  is  a  very  accurate  sugar  test. 

{b)  Feliling's  Test. — To  about  i  c.c.  of  FehHng's  solution^  in  a 
test-tube  add  about  4  c.c.  of  water,  and  boil.  This  is  done  to  deter- 
mine whether  the  solution  will  of  itself  cause  the  formation  of  a  pre- 
cipitate of  brownish-red  cuprous  oxide.  If  such  a  precipitate  forms, 
the  Fehling's  solution  must  not  be  used.  Add  sugar  solution  to  the 
warm  Fehling's  solution  a  few  drops  at  a  time  and  heat  the  mixture 
after  each  addition.  The  production  of  yellow  cuprous  hydroxide 
or  brownish-red  cuprous  oxide  indicates  that  reduction  has  taken  place. 
The  yellow  precipitate  is  more  likely  to  occur  if  the  sugar  solution  is 
added  rapidly  and  in  large  amount,  whereas  with  a  less  rapid  addition 
of  smaller  amounts  of  sugar  solution  the  brownish-red  precipitate  is 
generally  formed. 

This  is  a  much  more  satisfactory  test  than  Trommer's,  but  even  this 
test  is  not  entirely  reliable  when  used  to  detect  sugar  in  the  urine.  Such 
bodies  as  conjugate  glycuronates,  uric  acid,  nucleoprotein  and  homogen- 
tisic  acid  when  present  in  sufficient  amount  may  produce  a  result  sim- 
ilar to  that  produced  by  sugar.  Phosphates  of  the  alkaline  earths  may 
be  precipitated  by  the  alkali  of  the  Fehling's  solution  and  in  appearance 
may  be  mistaken  for  cuprous  hydroxide.  Cupric  hydroxide  may  also 
be  reduced  to  cuprous  oxide  and  this  in  turn  be  dissolved  by  creatinine, 
a  normal  urinary  constituent.  This  will  give  the  urine  under  examina- 
tion a  greenish  tinge  and  may  obscure  the  sugar  reaction  even  when  a 
considerable  amount  of  sugar  is  present. 

According  to  Laird^  even  small  amounts  of  creatinine  will  retard 
the  reaction  velocity  of  reducing  sugars  with  Fehling's  solution. 

*  Salkowski;  Zeil.  physiol.  Chem.,  79,  164,  1912. 

*  Fehling's  solution  is  composed  of  two  definite  solutions — a  copper  sulphate  solution  and 
an  alkaline  tartrate  solution,  which  may  be  prepared  as  follows: 

Copper  sulphate  solution  =34.65  grams  of  copper  sulphate  dissolved  in  water  and  made 
up  to  500  c.c. 

Alkaline  tartrate  solution  =125  grams  of  potassium  hydroxide  and  173  grams  of  Rochelle 
salt  dissolved  in  water  and  made  up  to  500  c.c. 

These  solutions  should  be  preserved  separately  in  rubber-stoppered  bottles  and  mixed 
in  equal  volumes  when  needed  for  use.     This  is  done  to  prevent  deterioration. 

'  Laird:  Journal  of  Pathology  and  Bacteriology,  16,  398,  1912. 


CARBOHYDRATES.  33 

(c)  Benedict's  Modifications  of  Fehling's  Test. — First  Modification. — 
To  2  c.c.  of  Benedict's  solution^  in  a  test-tube  add  6  c.c.  of  distilled 
water  and  7-9  drops  (not  more)  of  the  solution  under  examination. 
Boil  the  mixture  vigorously  for  about  15-30  seconds  and  permit  it  to 
cool  to  room  temperature  spontaneously.  (If  desired  this  process 
may  be  repeated,  although  it  is  ordinarily  unnecessary.)  If  sugar 
is  present  in  the  solution  a  precipitate  will  form  which  is  often  bluish- 
green  or  green  at  Tirst,  especially  if  the  percentage  of  sugar  is  low,  and 
which  usually  becomes  yellowish  upon  standing.  If  the  sugar  present 
exceeds  0.06  per  cent  this  precipitate  generally  forms  at  or  below  the 
boiling-point,  whereas  if  less  than  0.06  per  cent  of  sugar  is  present  the 
precipitate  forms  more  slowly  and  generally  only  after  the  solution  has 
cooled. 

Benedict  claims,  whereas  the  original  Fehling  test  will  not  serve 
to  detect  sugar  when  present  in  a  concentration  of  less  than  o.i  per 
cent,  that  the  above  modification  will  serve  to  detect  sugar  when 
present  in  as  small  cjuantity  as  0.015-0.02  per  cent.  Corroboration 
of  this  claim  of  increased  delicacy  has  recently  been  submitted  by 
Harrison.^ 

The  modified  Fehling  solution  used  in  the  above  test  differs  from 
the  original  Fehling  solution  in  that  100  grams  of  sodium  carbonate 
is  substituted  for  the  125  grams  of  potassium  hydroxide  ordinarily  used, 
thus  forming  a  Fehling  solution  which  is  considerably  less  alkaline 
than  the  original.  This  alteration  in  the  composition  of  the  Fehling 
solution  is  of  advantage  in  the  detection  of  sugar  in  the  urine  inasmuch 
as  the  strong  alkalinity  of  the  ordinary  Fehling  solution  has  a  tendency, 
when  the  reagent  is  boiled  with  a  urine  containing  a  small  amount  of 
dextrose,  to  decompose  sufficient  of  the  sugar  to  render  the  detection  of 
the  remaining  portion  exceedingly  difficult  by  the  usual  technic.  Bene- 
dict claims  that  for  this  reason  the  use  of  his  modified  solution  permits 
the  detection  of  much  smaller  amounts  of  sugar  than  does  the  use  of  the 
ordinary  Fehling  solution.  He  has  further  modified  his  solution  for 
use  in  the  quantitative  determination  of  sugar  (see  Chapter  XXII). 

Second  Modification.^ — Very  recently  Benedict  has  further  modi- 
fied his  solution  and  has  succeeded  in  obtaining  one  which  does  not 

'  Benedict's  modified  Fehling  solution  consists  of  two  definite  solutions — a  copper  sulphate 
solution  and  an  alkaline  tartrate  solution,  which  may  be  prepared  as  follows: 

Copper  sulphate  solution  =34.65  grams  of  copper  sulphate  dissolved  in  water  and  made 
up  to  500  c.c. 

Alkaline  tartrate  solution  =100  grams  of  anhydrous  sodium  carbonate  and  173  grams  of 
Rorhelle  salt  dissolved  in  water  and  made  up  to  500  c.c. 

These  solutions  should  be  preserved  separately  in  rubber-stoppered  bottles  and  mixed 
in  equal  volumes  when  needed  for  use.     This  is  done  to  prevent  deterioration. 

*  Harrison:  Pharnt.  Jour.,  87,  746,  igii. 

*  Benedict:  Jour.  Am.  Med.  Ass'n.,  57,  1193,  1911. 

3 


34  PHYSIOLOGICAL   CHEMISTRY. 

deteriorate  upon  long  standing.^  The  following  is  the  procedure  for 
the  detection  of  dextrose  in  solution :  To  five  cubic  centimeters  of  the 
reagent  in  a  test-tube  add  eight  (not  more)  drops  of  the  solution  under 
examination.  Boil  the  mixture  vigorously  for  from  one  to  two  minutes 
and  then  allow  the  fluid  to  cool  spontaneously.  In  the  presence  of  dex- 
trose the  entire  body  of  the  solution  will  he  filled  with  a  precipitate,  which 
may  be  red,  yellow  or  green  in  color,  depending  upon  the  amount  of  sugar 
present.  If  no  dextrose  is  present,  the  solution  will  remain  perfectly 
clear.  (If  urine  is  being  tested,  it  may  show  a  very  faint  turbidity,  due 
to  precipitated  urates.)  Even  very  small  quantities  of  dextrose  (o.i 
per  cent)  yield  precipitates  of  surprising  bulk  with  this  reagent,  and  the 
positive  reaction  for  dextrose  is  the  filling  of  the  entire  body  of  the  solu- 
tion with  a  precipitate,  so  that  the  solution  becomes  opaque.  Since 
amount  rather  than  color  of  the  precipitate  is  made  the  basis  of  this  test, 
it  may  be  applied  even  for  the  detection  of  small  quantities  of  dextrose, 
as  readily  in  artificial  light  as  in  daylight. 

{d)  Boettger^s  Test.- — To  5  c.c.  of  sugar  solution  in  a  test-tube  add 
I  c.c.  of  KOH  or  NaOH  and  a  very  small  amount  of  bismuth  subni- 
trate,  and  boil.  The  solution  will  gradually  darken  and  finally  as- 
sume a  black  color  due  to  reduced  bismuth.  If  the  test  is  made  on 
urine  containing  albumin  this  must  be  removed,  by  boiling  and  filtering, 
before  applying  the  test,  since  with  albumin  a  similar  change  of  color 
is  produced  (bismuth  sulphide). 

{e)  Nylander^s  Test  {Almens'  Test). — To  5  c.c.  of  sugar  solution 
in  a  test-tube  add  one-tenth  its  volume  of  Nylander's  reagent^  and  heat 
for  five  minutes  in  a  boiling  water-bath.^  The  solution  will  darken  if 
reducing  sugar  is  present,  and  upon  standing  for  a  few  moments  a  black 
color  will  appear.  This  color  is  due  to  the  precipitation  of  bismuth. 
If  the  test  is  made  on  urine  containing  albumin  this  must  be  removed, 
by  boiling  and  filtering,  before  applying  the  test,  since  with  albumin  a 
similar  change   of   color   is  produced.     Dextrose  when  present  to  the 

'  Benedict's  new  solution  has  the  following  composition: 

Copper  sulphate 17.3  grams. 

Sodium  citrate 1730  grams. 

Sodium  carbonate  (anhydrous) 100. o  grams. 

Distilled  water  to  make  i  liter. 
With  the  aid  of  heat  dissolve  the  sodium  citrate  and  carbonate  in  about  600  c.c.  of  water. 
Pour  (through  a  folded  filter  paper  if  necessary)  into-a  glass  graduate  and  make  up  to  850  c.c. 
Dissolve  the  copper  sulphate  in  about  100  c.c.  of  water  and  make  up  to  150  c.c.  Pour  the 
carbonate-citrate  solution  into  a  large  beaker  or  casserole  and  add  the  copper  sulphate  solu- 
tion slowly,  with  constant  stirring.  The  mixed  solution  is  ready  for  use  and  does  not  deterio- 
rate upon  long  standing. 

-  Nylander's  reagent  is  prepared  by  digesting  2  grams  of  bismuth  subnitrate  and  4  grams 
of  Rochelle  salt  in  100  c.c  of  a  10  per  cent  potassium  hydroxide  solution.  The  reagent  is 
then  cooled  and  filtered. 

'  Hammarsten  suggests  that  the  mixture  should  be  boiled  2-5  minutes  (according  to  the 
sugar  content)  over  a  free  flame  and  the  tube  then  permitted  to  stand  5  minutes  before 
drawing  conclusions. 


CARBOHYDRATES. 


35 


extent  of  0.08  per  cent  may  be  detected  by  this  reaction.  It  is  claimed 
by  Bech'old  that  Nylander's  and  Boettger's  tests  give  a  negative  reaction 
with  solutions  containing  sugar  when  mercuric  chloride  or  chloroform 
is  present.  Other  observers^  have  failed  to  verify  the  inhibitory  action 
of  mercuric  chloride  and  have  shown  that  the  inhibitory  influence  of 
chloroform  may  be  overcome  by  raising  the  temperature  of  the  urine 
to  the  boiling-point  for  a  period  of  five  minutes  previous  to  making  the 
test.  Urines  rich  in  indican,  urochrome,  nroerythrin  or  hcEmatopor phyrin, 
as  well  as  urines  excreted  after  the  ingestion  of  large  amounts  of  certain 
medicinal  substances,  may  give  a  darkening  of  Nylander's  reagent  similar 
to  that  of  a  true  sugar  reaction.  It  is  a  disputed  point  whether  the  urine 
after  the  administration  of  urotropin  will  reduce  Nylander's  reagent.^ 
Strausz'  has  recently  shown  that  the  urine  of  diabetics  to  whom  ''  lothion  " 
(diiodohydroxypropane)  has  been  administered  will  give  a  negative 
Nylander's  reaction  and  respond  positively  to  the  Fehling  and  polari- 
scopic  tests.  "lothion"  also  interferes  with  the  Nylander  test  w  ^'//ro 
whereas  KI  and  I  do  not. 

According  to  Rustin  and  Otto,  the  addition  of  PtCl^  increases  the 
delicacy  of  Nylander's  reaction.  They  claim  that  this  procedure  causes 
the  sugar  to  be  converted  quantitatively.  No  quantitative  method  has 
yet  been  devised,  however,  based  upon  this  principle. 

Bohmansson*  before  testing  the  urine  under  examination  treats 
it  (10  c.c.)  with  1/5  volume  of  25  per  cent  hydrochloric  acid  and  about 
1/2  volume  of  bone  black.  This  mixture  is  shaken  one  minute,  then 
filtered  and  the  neutralized  filtrate  tested  by  Nylander's  reaction.  Boh- 
mansson  claims  that  this  procedure  removes  certain  interfering  substances, 
in  particular  urochrome. 

A  positive  Nylander  or  Boettger  test  is  probably  due  to  the  following 
reactions: 

(a)     Bi  (OH)  2NO3  +  KOH— Bi(OH)  3  +  KNO3. 
{b)  2Bi(OH)3-30--Bi3  +  3H30. 

12.  Fermentation  Test. — "Rub  up"  in  a  mortar  about  20  c.c. 
of  the  sugar  solution  with  a  small  piece  of  compressed  yeast.  Transfer 
the  mixture  to  a  saccharometer  (shown  in  Fig.  3,  p.  36)  and  stand  it  aside 
in  a  warm  place  for  about  twelve  hours.  If  the  sugar  is  fermentable, 
alcoholic  fermentation  will  occur  and  carbon  dioxide  will  collect  as  a 
gas  in  the  upper  portion  of  the  tube.     On  the  completion  of  fermenta- 

^  Kehixxss  a.nd}i&\\\!i;  Journal  of  Biological  Chemistry,  -J,  26-],   1910;  also  Zeidlitz;  Upsala 
Lakdreforen  Fork.,  N.  F.,  11,  igo6. 

^  Abt;  Archives  oj  Pediatrics,  24,  275,    1907;   also  Weitbrecht;  Schweiz.   Wochschr.,    47, 

577>  1909- 

'  Strausz;  Miinch.  med.  Woch.,  59,  85,  1912. 
*  Bohmansson:  Biochem.  Zeit.,  19,  p.  281. 


36 


PHYSIOLOGICAL   CHEMISTRY. 


tion  introduce  a  little  potassium  hydroxide  solution  into  the  graduated 
portion  by  means  of  a  bent  pipette,  place  the  thumb  tightly  over  the  open- 
ing in  the  apparatus  and  invert  the  saccharometer.  Explain  the  result. 
The  important  findings  of  Neuberg  and  associates^  recently  re- 
ported indicate  very  clearly  that  the  liberation  of  carbon  dioxide  by  yeast 
is  not  necessarily  a  criterion  of  the  presence  of  sugar.  The  presence 
of  a  new  enzyme  called  carboxylase  has  been 
demonstrated  in  yeast  which  has  the  power  of 
splitting  off  CO  ^  from  the  carhoxyl  group  of  amino 
and  other  aliphatic  acids. 

13.  Barfoed's  Test. — Place  about  5  c.c.  of 
Barfoed's  solution^  in  a  test-tube  and  heat  to 
boiling.  Add  dextrose  solution  slowly,  a  few 
drops  at  a  time,  heating  after  each  addition. 
Reduction  is  indicated  by  the  formation  of  a 
red  precipitate.  If  the  precipitate  does  not  form 
upon  continued  boiling  allow  the  tube  to  stand  a 
few  minutes  and  examine.  Sodium  chloride 
interferes  with  the  reaction  (Welker). 

Barfoed's  test  is  not  a  specific  test  for  dextrose 
as  is  frequently  stated,  but  simply  serves  to 
detect  monosaccharides.  Disaccharides  will  also 
respond  to  the  test,  under  proper  conditions  of 
acidity.^  Also  if  the  sugar  solution  is  boiled 
sufl&ciently  long,  in  contact  with  the  reagent,  to 
hydrolyze  the  disaccharide  through  the  action  of  the  acetic  acid  present 
in  the  Barfoed's  solution  a  positive  test  results.* 

14.  Formation  of  Caramel. — Gently  heat  a  small  amount  of  pul- 
verized dextrose  in  a  test-tube.  After  the  sugar  has  melted  and  turned 
brown,  allow  the  tube  to  cool,  add  water  and  warm.  The  coloring 
matter  produced  is  known  as  caramel. 

15.  Demonstration  of  Optical  Activity. — A  demonstration  of  the 
use  of  the  polariscope,  by  the  instructor,  each  student  being  required 
to  take  readings  and  compute  the  "specific  rotation." 


Fig.  3. — EixHORN  Sac- 
charometer. 


Use  of  the  Polariscope. 

For  a  detailed  description  of  the  different  forms  of  polariscopes,  the 
method   of   manipulation   and  the  principles  involved,  the  student  is 

'Neuberg  and  Associates:   Biochem.  Zeitsch.,  31,  170;  32,  323;  36,  (60,  68,  76),  1911. 
'  Barfoed's  solution  is  prepared  as  follows:     Dissolve  4.5  grams  of  neutral  crystallized 
copper  acetate  in  100  c.c.  of  water  and  add  1.2  c.c.  of  50  per  cent  acetic  acid. 

*  Mathews  and  McGuigan:  Am.  Jour.  Physiol.,  19,  175,  1907. 

*  Hinkle  and  Sherman:  Jour.  Am.  Chem.Soc,  29,  1744,  1907. 


CARBOHYDRATES. 


37 


referred  to  any  standard  text-book  of  physics.  A  brief  description  fol- 
lows: An  ordinary  ray  of  light  vibrates  in  every  direction.  If  such  a  ray 
is  caused  to  pass  through  a  "polarizing"  Nicol  prism  it  is  resolved  into 
two  rays,  one  of  which  vibrates  in  every  direction  as  before  and  a  second 
ray  which  vibrates  in  one  plane  only.  This  latter  ray  is  said  to  be  polar- 
ized. Many  organic  substances  (sugars,  proteins,  etc.)  have  the  power 
of  twisting  or  rotating  this  plane  of  polarized  light,  the  extent  to  which 
the  plane  is  rotated  depending  upon  the  number  of  molecules  which 


Fig.  4. — One  Form  of  Laurent  Polariscope. 

B,  Microscope  for  reading  the  scale;  C,  a  vernier;  E,  position  of  the  analyzing  Nicol  prism; 

H,  polarizing  Nicol  prism  in  the  tube  below  this  point. 


the  polarized  light  passes.  Substances  which  possess  this  power  are 
said  to  be  "optically  active."  The  specific  rotalmi  of  a  substance  is  the 
rotation  expressed  in  degrees  which  is  afforded  by  one  gram  of  substance 
dissolved  in  i  c.c.  of  water  in  a  tube  one  decimeter  in  length.  The  spe- 
cific rotation,  (a)^,  may  be  calculated  by  means  of  the  following  formula, 

p.l 

in  which 

£>=;=  sodium  light. 

a  =  observed  rotation  in  degrees. 

/»  =  grams  of  substance  dissolved  in  i  c.c.  of  liquid. 

/==  length  of  the  tube  in  decimeters. 
If  the  specific  rotation  has  been  determined  and  it  is  desired  to  ascertain 
the  per  cent  of  the  substance  in  solution,  this  may  be  obtained  by  the 
use  of  the  following  formula. 


P  = 


(«)d  I 


38 


PHYSIOLOGICAL    CHEMISTRY. 


The  value  of  p  multiplied  by  loo  will  be  the  percentage  of  the  sub- 
stance in  solution. 

An  instrument  by  means  of  which  the  extent  of  the  rotation  may 
be  determined  is  called  a  polariscope  or  polarimeter.     Such  an  instru- 


Kini 


Fig.    V — D1AGRAMM.A.TIC  Representatiox  of  the  Course  of  the  Light  through  the 

Laurent  Polariscope.     (The  direction  is  reversed  from  that  of  Fig.  4,  p.  37.) 

a,  Bichromate  plate  to  purify  the  light;  b,  the  polarizing  Nicol  prism;  c,  a  thin  quartz 

plate  covering  one-half  the  field  and  essential  in  producing  a  second  polarized  plane;  d,  tube 

to  contain  the  liquid  under  examination;  e,  the  analyzing  Nicol  prism; /and  g,  ocular  lenses. 

ment  designed  especially  for  the  examination  of  sugar  solutions  is  termed 
a  saccharimeter  or  polarizing  saccharimeter.  The  form  of  polariscope 
in  Fig.  4,  p.  37,  consists  essentially  of  a  long  barrel  provided  with  a 


Fig.  6. — Polariscope  (Schmidt  and  Haensch  Model). 

Nicol  prism  at  either  end  (Fig.  5 ,  above) .  The  solution  under  examination 
is  contained  in  a  tube  which  is  placed  between  these  two  prisms.  At 
the  front  end  of  the  instrument  is  an  adjusting  eyepiece  for  focusing  and 


CARBOHYDRATES.  39 

a  large  recording  disc  which  registers  in  degrees  and  fractions  of  a  degree. 
The  light  is  admitted  into  the  far  end  of  the  instrument  and  is  ])ohirizcd 
by  passing  through  a  Nicol  ])rism.  This  polari^^cd  ray  then  traverses  the 
column  of  liquid  within  the  tube  mentioned  above  and  if  the  substance 
is  optically  active  the  plane  of  the  polarized  ray  is  rotated  to  the  right  or 
left.  Bodies  rotating  the  ray  to  the  right  are  called  dextro-rotatory 
and  those  rotating  it  to  the  left  I cevo -rotatory. 

Within  the  apparatus  is  a  disc  which  is  so  arranged  as  to  be  without 
lines  and  uniformly  light  at  zero.  Upon  placing  the  optically  active 
substance  in  position,  however,  the  plane  of  polarized  light  is  rotated 
or  turned  and  it  is  necessary  to  rotate  the  disc  through  a  certain  number 
of  degrees  in  order  to  secure  the  normal  conditions,  i.  e.,  ''without  lines 
and  uniformly  light."  The  difference  between  this  reading  and  the 
zero  is  a  or  the  observed  rotation  in  degrees. 

Polarizing  saccharimeters  are  also  constructed  by  which  the  per- 
centage of  sugar  in  solution  is  determined  by  making  an  observation 
and  multiplying  the  value  of  each  division  on  a  horizontal  sliding  scale 
by  the  value  of  the  division  expressed  in  terms  of  dextrose.  This  factor 
may  vary  according  to  the  instrument. 

"Optical"  methods  embracing  the  determination  of  the  optical 
activity  are  being  utilized  in  recent  years  in  many  "quantitative" 
connections.^ 

CH^OH 
L^EVULOSE,  (CH0H)3. 
CO 

1 

CH,OH 

As  already  stated,  laevulose,  sometimes  called  fructose  or  fruit  sugar, 
occurs  widely  disseminated  throughout  the  plant  kingdom  in  company 
with  dextrose.  Its  reducing  power  is  somewhat  weaker  than  that  of 
dextrose.  Laevulose  does  not  ordinarily  occur  in  the  urine  in  diabetes 
mellitus,  but  has  been  found  in  exceptional  cases.  With  phenylhydrazine 
it  forms  the  same  osazone  as  dextrose.  With  methylphenylhydrazine, 
laevulose  forms  a  characteristic  methylphenyllaev^losazone. 

(For  a  further  discussion  of  laevulose  see  the  section  on  Hexoses, 
p.  26.) 

I  Abderhalden  and  Schmidt:  "Determination  of  blood  content  by  means  of  the  optica 
method,"  Zeit.  physiol.  Chem.  66,  120,  1910;  also  C.  Neuberg;  "Determination  of  nucleic  acid 
cleavage  by  polarization,"  Biodiemische  Zeitschrijt,  30,  505,  191 1 


40  physiological  chemistry. 

Experiments  on  L.evulose. 

1-13.  Repeat  these  experiments  as  given  under  Dextrose,  pages 
27-36. 

14.  Seliwanoff's  Reaction. — To  5  c.c'  of  Seliwanoff's  reagent^ 
in  a  test-tube  add  a  few  drops  of  a  Isvulose  solution  and  heat  the  mix- 
ture to  boiling.  A  positive  reaction  is  indicated  by  the  production 
of  a  red  color  and  the  separation  of  a  red  precipitate.  The  latter  may 
be  dissolved  in  alcohol  to  which  it  will  impart  a  striking  red  color. 

If  the  boiling  be  prolonged  a  similar  reaction  may  be  obtained  with 
solutions  of  dextrose  or  maltose.  This  has  been  explained^  in  the  case 
of  dextrose  as  due  to  the  transformation  of  the  dextrose  into  laevulose  by  the 
catalytic  action  of  the  hydrochloric  acid.  The  precautions  necessary  for  a 
positive  test  for  laevulose  are  as  follows:  The  concentration  of  the 
hydrochloric  acid  must  not  be  more  than  12  per  cent.  The  reaction 
(red  color),  and  the  precipitate  must  be  observed  after  not  more  than 
20-30  seconds  boiling.  Dextrose  must  not  be  present  in  amounts  ex- 
ceeding 2  per  cent.  The  precipitate  must  be  soluble  in  alcohol  with  a 
bright  red  color. 

15.  Borchardt's  Reaction. — To  about  5  c.c.  of  a  solution  of  laevulose 
in  a  test-tube  add  an  equal  volume  of  25  per  cent  hydrochloric  acid  and  a 
few  crystals  of  resorcinol.  Heat  to  boiling  and  after  the  production  of  a 
red  color,  cool  the  tube  under  running  water  and  transfer  to  an  evapo- 
rating dish  or  beaker.  Make  the  mixture  slightly  alkaline  with  solid 
potassium  hydroxide,  return  it  to  a  test-tube,  add  2-3  c.c.  of  acetic  ether 
and  shake  the  tube  vigorously.  In  the  presence  of  lae\Talose,  the 
acetic  ether  is  colored  yellow.  (For  further  discussion  of  the  test  see 
Chapter  XIX.) 

16.  Formation  of  Methylphenyllaevulosazone. — To  a  solution 
of  1.8  grams  of  laevulose  in  10  c.c.  of  water  add  4  grams^  of  methyl- 
phenylhydrazine  and  enough  alcohol  to  clarify  the  solution.  Intro- 
duce 4  c.c.  of  50  per  cent  acetic  acid  and  heat  the  mixture  for  5-10  min- 
utes on  a  boiling  water-bath.^  On  standing  15  minutes  at  room  tem- 
perature, crystallization  begins  and  is  complete  in  two  hours.  By  scratch- 
ing the  sides  of  the  flask  or  by  inoculation,  the  solution  quickly  con- 
geals to  form  a  thick  paste  of  reddish-yellow  silky  needles.  These  are 
the  crystals  of  inethylphenyllcBVulosazone.  They  may  be  recrystallized 
from  hot  95  per  cent  alcohol  and  melt  at  153°  C. 

*  Seliwanoff's  reagent  may  be  prepared  by  dissolving  0.05  gram  of  resorcinol  in  100  c.c. 
of  dilute  (1:2)  hydrochloric  acid. 

^Koenigsfeld:  Bioch.'Zeit.,  38,  311,  19 12 
'3.66  grams  if  absolutely  pure. 

*  Longer  heating  is  to  be  avoided. 


CARBOHYDRATES.  4I 

CH.OH 
GALACTOSE,  (CHOH),. 

CHO 

Galactose  occurs  with  dextrose  as  one  of  the  products  of  ihe  hydro- 
lysis of  lactose.  It  is  dextro-rotatory,  forms  an  osazone  with  j)hcnyl- 
hydrazine  and  ■ferments  slowly  with  yeast.  Upon  oxidation  with  nitric 
acid  galactose  yields  mucic  acid,  thus  differentiating  this  monosac- 
charide from  dextrose  and  laevulose.  Lactose  also  yields  mucic  acid 
under  these  conditions.  The  mucic  acid  test  may  be  used  in  urine 
examination  to  differentiate  lactose  and  galactose  from  other  reducing 
sugars.     The  assimilation  limit  for  galactose  is  30-40  grams.  ^ 

Experiments  on  Galactose. 

1.  Tollens'  Reaction. — To  equal  volumes  of  galactose  solution  and 
hydrochloric  acid  (sp.  gr.  1.09)  add  a  little  phloroglucinol,  and  heat  the 
mixture  on  a  boiling  water-bath.  Galactose,  pentose  and  glycuronic 
acid  will  be  indicated  by  the  appearance  of  a  red  color.  Galactose 
may  be  differentiated  from  the  two  latter  substances  in  that  its  solutions 
exhibit  no  absorption  bands  upon  spectroscopical  examination. 

2.  Mucic  Acid  Test. — Treat  100  c.c.  of  the  solution  containing 
galactose  with  20  c.c.  of  concentrated  nitric  acid  (sp.  gr.  1.4)  and  evapo- 
rate the  mixture  in  a  broad,  shallow  glass  vessel  on  a  boiling  water- 
bath  until  the  volume  of  the  mixture  has  been  reduced  to  about  20  c.c. 
At  this  point  the  fluid  should  be  clear,  and  a  fine  white  precipitate  of 
mucic  acid  should  form.  If  the  percentage  of  galactose  present  is  low 
it  may  be  necessary  to  cool  the  solution  and  permit  it  to  stand  for  some 
time  before  the  precipitate  will  form.  It  is  impossible  to  differentiate 
between  galactose  and  lactose  by  this  test,  but  the  reaction  serves  to 
differentiate  these  two  sugars  from  all  other  reducing  sugars.  Differ- 
entiate lactose  from  galactose  by  means  of  Barfoed's  test  (p.  36). 

3.  Phenylhydrazine  Reaction. — Make  the  test  according  to  direc- 
tions given  under  Dextrose,  3  or  4,  pages  28  and  29. 

Pentoses,  C.HjoOg. 

In  plants  and  more  particularly  in  certain  gums,  very  complex  car- 
bohydrates, called  pentosans  (see  p.  55),  occur.  These  pentosans  through 
hydrolysis  by  acids  may  be  transformed  into  pentoses.  Pentoses  do  not 
ordinarily  occur  in  the  animal  organism,  but  have  been  found  in  the 

"Brasch:  Zeitschrift  fur  Biologic,  50,  113,  1907. 


42  PHYSIOLOGICAL    CHEMISTRY. 

urine  of  morphine  habitues  and  others,  their  occurrence  sometimes 
being  a  persistent  condition  without  known  cause.  They  may  be  ob- 
tained from  the  hydrolysis  of  nucleoproteins  being  present  in  the  nucleic 
acid  complex  of  the  molecule.  Pentoses  are  non-fermentable,  have 
strong  reducing  power  and  form  osazones  with  phenylhydrazine.  Pen- 
toses are  an  important  constituent  of  the  dietary  of  herbivorous  animals. 
Glycogen  is  said  to  be  formed  after  the  ingestion  of  these  sugars  containing 
five  oxygen  atoms.  This,  however,  has  not  been  conclusively  proven. 
On  distillation  with  strong  hydrochloric  acid  pentoses  and  pentosans 
yield  furfurol,  which  can  be  detected  by  its  characteristic  red  reaction 
with  aniline-acetate  paper. 

CH^OH 

ARABINOSE,   (CHOHjg. 
CHO 

Arabinose  is  one  of  the  most  important  of  the  pentoses.  The  l- 
arabinose  may  be  obtained  from  gum  arabic,  plum  or  cherry  gum.  by 
boiling  for  lo  minutes  with  concentrated  hydrochloric  acid.  This  pentose 
is  dextro-rotatory,  forms  an  osazone  and  has  reducing  power,  but  does  not 
ferment.  The  ^'-arabinose  has  been  isolated  from  the  urine  and  yields 
an  osazone  which  melts  at  i66°-i68°  C. 

Experiments  on  Arabinose. 

1.  Bial's  Reaction/— To  5  c.c.  of  Bial's  reagent^  in  a  test-tube 
add  2-3  c.c.  of  the  arabinose  solution  and  heat  the  mixture  gently  until 
the  first  bubbles  rise  to  the  surface.  Immediately  or  upon  cooling  the 
solution  becomes  green  and  a  flocculent  precipitate  of  the  same  color 
may  form.  (For  further  discussion  see  Chapter  XIX) .  The  test  may  also 
be  performed  by  adding  the  pentose  to  the  hot  reagent. 

2.  ToUens'  Reaction.— To  equal  volumes  of  arabinose  solution 
and  hydrochloric  acid  (sp.  gr.  1.09)  add  a  little  phloroglucinol  and  heat 
the  mixture  on  a  boiling  water-bath.  Galactose,  pentose  or  glycuronic 
acid  will  be  indicated  by  the  appearance  of  a  red  color.  To  differentiate 
between  these  bodies  make  a  spectroscopic  examination  and  look  for 
the  absorption  band  between  D  and  E  given  by  pentoses  and  glycuronic 
acid.  Differentiate  between  the  two  latter  bodies  by  the  melting-points 
of  their  osazones. 

'  Bial:  Deut.  med.  Woch.,  28,  252,  1902. 
^  Orcinol 1.5  gram. 

Fuming  HCl 500  grams. 

Ferric  chloride  (10  per  cent) .  .20-30  drops. 


CARBOHYDRATES.  43 

Compare  the  reaction  with  that  obtained  with  galactose  (page  41). 

3.  Orcinol  Test. — Repeat  i,  using  orcinol  instead  of  phloroglucinol. 
A  succession  of  colors  from  red  through  reddish-blue  to  green  is  pro- 
duced. A  green  precipitate  is  formed  which  is  soluble  in  amyl  alcohol 
and  has  absorption  Ijands  between  C  and  D. 

4.  Phenylhydrazine  Reaction. — Make  this  test  on  the  arabinose 
solution  according  to  directions  given  under  Dextrose,  3  or  4,  pages 
28  and  29. 

CH2OH 
XYLOSE,  (CHOH)3. 

CHO 

Xylose,  or  wood  sugar,  is  obtained  by  boiling  wood  gums  with  di- 
lute acids  as  explained  under  Arabinose,  page  43.  It  is  dextro-rota- 
tory, forms  an  osazone  and  has  reducing  power,  but  does  not  ferment. 

Experiments  on  Xylose. 
1-4.  Same  as  for  arabinose  (see  above). 

RHAMNOSE,  C«H,,0.. 

'  6        12        5 

Rhamnose  or  methyl-pentose  is  an  example  of  a  true  carbohydrate 
which  does  not  have  the  H  and  O  atoms  present  in  the  proportion  to 
form  water.     Its  formula  is  CpH,  ,0..     It  has  been  found  that  rham- 

C        12       o 

nose  when  ingested  by  rabbits  or  hens  has  a  positive  influence  upon  the 
formation  of  glycogen  in  those  organisms. 

DISACCHARIDES,  C.^H^^O^^. 

The  disaccharides  as  a  class  may  be  divided  into  two  rather  dis- 
tinct groups.  The  first  group  would  include  those  disaccharides  which 
are  found  in  nature  as  such,  e.  g.,  sucrose  and  lactose  and  the  second 
group  would  include  those  disaccharides  formed  in  the  hydrolysis  of  more 
complex  carbohydrates,  e.  g.,  maltose,  and  iso-maltose. 

The  disaccharides  have  the  general  formula  C^^'B.^Jd^^,  to  which, 
in  the  process  of  hydrolysis,  a  molecule  of  water  is  added  causing  the 
single  disaccharide  molecule  to  split  into  two  monosaccharide  (hexose) 
molecules.  The  products  of  the  hydrolysis  of  the  more  common  dis- 
accharides are  as  follows: 

Maltose  =  dextrose  -1-  dextrose. 
Lactose  =  dextrose  +  galactose. 
Sucrose  =  dextrose  -|-  laevulose. 


44  PHYSIOLOGICAL    CHEMISTRY. 

All  of  the  more  common  disaccharides  except  sucrose  have  the  power 
of  reducing  certain  metallic  oxides  in  alkaline  solution,  notably  those 
of  copper  and  bismuth.  This  reducing  power  is  due  to  the  presence 
of  the  aldehyde  group  ( — CHO)  in  the  sugar  molecule. 

MALTOSE,  C^^H^^O,,. 

Maltose  or  malt  sugar  is  formed  in  the  hydrolysis  of  starch  through 
the  action  of  an  enzyme,  vegetable  amylase  {diastase),  contained  in  sprout- 
ing barley  or  malt.  Certain  enzymes  in  the  saliva  and  in  the  pancreatic 
juice  may  also  cause  a  similar  hydrolysis.  Maltose  is  also  an  intermediate 
product  of  the  action  of  dilute  mineral  acids  upon  starch.  It  is  strongly 
dextro-rotatory,  reduces  metallic  oxides  in  alkaline  solution  and  is  fer- 
mentable by  yeast  after  being  inverted  (see  Polysaccharides,  page  47) 
by  the  enzyme  maltase  of  the  yeast.  In  common  with  the  other  disac- 
charides, maltose  may  be  hydrolyzed  with  the  formation  of  two  molecules 
of  monosaccharide.  In  this  instance  the  products  are  two  molecules  of 
dextrose.  With  phenylhydrazine  maltose  forms  an  osazone,  mallo- 
sazone.  The  following  formula  represents  the  probable  structure  of 
maltose: 

CH2OH  CHO 

I  I 

CHOH  CHOH 

CHO —  CHOH 

I  I 

CHOH  CHOH 

CHOH  CHOH 

C-^^ O       CH2 

\ 

H 

Maltose. 

Expp:riments  on  Maltose. 
1-13.  Repeat  these  experiments  as  given  under  Dextrose,  pages  27-36. 

ISO-MALTOSE,  C^^^fi^^. 

Iso-maltose,  an  isomeric  form  of  maltose;  is  formed,  along  with  maltose, 
by  the  action  of  diastase  upon  starch  paste,  and  also  by  the  action  of  hydro- 
chloric acid  upon  dextrose.  It  also  occurs  with  maltose  as  one  of  the 
products  of  salivary  digestion.  It  is  dextro-rotatory  and  with  phenylhy- 
drazine gives  an  osazone  which  is  characteristic.     Iso-maltose  is  very 


CARBOHYDRATES.  45 

soluble  and  reduces  the  oxides  of  bismuth  and  copper  in  alkaline  solution. 
Pure  iso-maltose  is  probably  only  slightly  fermentable. 

LACTOSE,  Cj,H,,0,,. 

Lactose  or  milk  sugar  occurs  ordinarily  only  in  milk,  but  has  often 
been  found  in  the  urine  of  women  during  pregnancy  and  lactation.  It 
may  also  occur  -in  the  urine  of  normal  persons  after  the  ingestion  of 
unusually  large  amounts  of  lactose  in  the  food.  It  has  a  strong  reducing 
power,  is  dextro-rotatory  and  forms  an  osazone  with  phenylhydrazine. 
Upon  hydrolysis  lactose  yields  one  molecule  of  dextrose  and  one  molecule 
of  galactose. 

In  the  souring  of  milk  the  bacterium  laclis  and  certain  other  micro- 
organisms bring  about  lactic  acid  fermentation  by  transforming  the  lac- 
tose of  the  milk  into  lactic  acid, 

H        OH 
H— C  —  C— COOH, 
H        H 

and  alcohol.  This  same  reaction  may  occur  in  the  alimentary  canal  as 
the  result  of  the  action  of  putrefactive  bacteria.  In  the  preparation  of 
kephyr  and  koumyss  the  lactose  of  the  milk  undergoes  alcoholic  fermenta- 
tion, through  the  action  of  ferments  other  than  yeast,  and  at  the  same 
time  lactic  acid  is  produced.  Lactose  and  galactose  yield  mucic  acid  on 
oxidation  with  nitric  acid.  This  fact  is  made  use  of  in  urine  analysis  to 
facilitate  the  differentiation  of  these  sugars  from  other  reducing  sugars. 
Lactose  is  not  fermentable  by  pure  yeast. 

Experiments  on  Lactose. 

1-13.  Repeat  these  experiments  as  given  under  Dextrose,  pages  27-36. 

14.  Mucic  Acid  Test. — Treat  100  c.c.  of  the  solution  containing 
lactose  with  20  c.c.  of  concentrated  nitric  acid  (sp.  gr.  1.4)  and  evaporate 
the  mixture  in  a  broad,  shallow  glass  vessel  on  a  boiling  water-bath,  until 
the  volume  of  the  mixture  has  been  reduced  to  about  20  c.c.  At  this 
point  the  fluid  should  be  clear,  and  a  fine  white  precipitate  of  mucic  acid 
should  form.  If  the  percentage  of  lactose  present  is  low  it  may  be  neces- 
sary to  cool  the  solution  and  permit  it  to  stand  for  some  time  before  the  pre- 
cipitate will  appear.  It  is  impossible  to  differentiate  between  lactose 
and  galactose  by  this  test,  but  the  reaction  serves  to  differentiate  these 
two  sugars  from  all  other  reducing  sugars. 


46 


PHYSIOLOGICAL   CHEMISTRY. 


Differentiate  lactose  from  galactose  by  means  of  Barfoed's  test, 
page  36. 

SUCROSE,  C,,H,20,,. 

Sucrose,  also  called  saccharose  or  cane  sugar,  is  one  of  the  most 
important  of  the  sugars  and  occurs  very  extensively  distributed  in  plants, 
particularly  in  the  sugar  cane,  sugar  beet,  sugar  millet  and  in  certain 
palms  and  maples,  ^ 

Sucrose  is  dextro-rotatory  and  upon  hydrolysis,  as  before  mentioned, 
the  molecule  of  sucrose  takes  on  a  molecule  of  water  and  breaks  down 
into  two  molecules  of  monosaccharide.  The  monosaccharides  formed  in 
this  instance  are  dextrose  and  laevulose.     This  is  the  reaction: 

C12H22O11  +  H2O— CeH^20g+CgH^20( 


Sucrose. 


Dextrose. 


Laevulose. 


This  process  is  called  inversion  and  may  be  produced  by  bacteria,  enzymes, 
and  certain  weak  acids.  After  this  inversion  the  previously  strongly 
dextro-rotatory  solution  becomes  laevo-rotatory.  This  is  due  to  the  fact 
that  the  laevulose  molecule  is  more  strongly  laevo-rotatory  than  the  dex- 
trose molecule  is  dextro-rotatory.  The  product  of  this  inversion  is  called 
invert  sugar. 

Sucrose  does  not  reduce  metallic  oxides  in  alkaline  solution  and  forms 
no  osazone  with  phenylhydrazine.  It  is  not  fermentable  directly  by  yeast, 
but  must  first  be  inverted  by  the  enzyme  sucrase  {invertase  or  invertin) 
contained  in  the  yeast.  The  probable  structure  of  sucrose  may  be  repre- 
sented by  the  following  formula.  Note  the  absence  of  any  free  ketone 
or  aldehyde  group. 


CH2OH 


CHOH 


CH2OH 


CHO 


Sucrose. 

Experiments  on  Sucrose. 


1-13,  Repeat  these  experiments  according  to  the  directions  given 
under  Dextrose,  pages  27-36. 


CARBOHYDRiVTES. 


47 


14.  Inversion  of  Sucrose. — To  25  c.c.  of  sucrose  solution  in  a 
beaker  add  5  drops  of  concentrated  HCl  and  boil  one  minute.  Cool 
the  solution,  render  alkaline  with  solid  KOH  and  upon  the  resulting  fluid 
repeat  experiments  3  (or  4)  and  11  as  given  under  Dextrose,  pages  28-30. 
Explain  the  results. 

15.  Production  of  Alcohol  by  Fermentation. — Prepare  a  strong 
(io-2o  per  cent)  solution  of  sucrose,  add  a  small  amount  of  egg  albumin 
or  commercial  peptone  and  introduce  the  mixture  into  a  bottle  of  appro- 
priate size.  Add  yeast,  and  by  means  of  a 
bent  tube  inserted  through  a  stopper  into  the 
neck  of  the  bottle,  conduct  the  escaping  gas 
into  water.  As  fermentation  proceeds  readily 
in  a  warm  place  the  escaping  gas  may  be 
collected  in  a  eudiometer  tube  and  examined. 
When  the  activity  of  the  yeast  has  practically 
ceased,  filter  the  contents  of  the  bottle  into  a 
flask  and  distil  the  mixture.  Catch  the  first 
portion  of  the  distillate   separately  and  test 

-,-,,.  .  Fig.  7. — Iodoform.  {Autenneth.) 

for  alcohol  by  one  of  the  following  reactions : 

(a)  Iodoform  Test. — Render  2-3  c.c.  of  the  distillate  alkaline  with 
potassium  hydroxide  solution  and  add  a  few  drops  of  iodine  solution. 
Heat  gently  and  note  the  formation  of  iodoform  crystals.  Examine  these 
crystals  under  the  microscope  and  compare  them  with  those  in  Fig.  7. 

(b)  Aldehyde  Test. — Place  5  c.c.  of  the  distillate  in  a  test-tube,  add  a 
few  drops  of  potassium  dichromate  solution,  K^CrjO^,  and  render  it  acid 
with  dilute  sulphuric  acid.  Boil  the  acid  solution  and  note  the  odor  of 
aldehyde. 

TRISACCHARIDES,  CjgHg.Oie. 

RAFFINOSE. 

This  trisaccharide,  also  called  melitose,  or  melitriose  occurs  in  cotton 
seed,  Australian  manna,  and  in  the  molasses  from  the  preparation  of 
beet  sugar.  It  is  dextro-rotatory,  does  not  reduce  Fehling's  solution,  and 
is  only  partly  fermentable  by  yeast. 

Raffinose  may  be  hydrolyzed  by  weak  acids  the  same  as  the  poly- 
saccharides are  hydrolyzed,  the  products  being  lasvulose  and  melibiose; 
further  hydrolysis  of  the  melibiose  yields  dextrose  and  galactose. 

POLYSACCHARIDES,  (C,'ii,,0,)^. 

In  general  the  polysaccharides  are  amorphous  bodies,  a  few,  how- 
ever,  are   crystallizable.     Through   the   action   of  certain   enzvmes  or 


48  PHYSIOLOGICAL   CHEMISTRY. 

weak  acids  the  polysaccharides  may  be  hydrolyzed  with  the  formation  of 
monosaccharides.  As  a  class  the  polysaccharides  are  quite  insoluble  and 
are  non-fermentable  until  inverted.  By  inversion  is  meant  the  hydrolysis 
of  disaccharide  or  polysaccharide  sugars  to  form  monosaccharides,  as 
indicated  in  the  following  equations: 

(a)  C,3H,30,,H-H30-2(C,H,,Oe). 

(b)  C«H,,0,  +  H,0-CeH,30,. 

STARCH,   (CeH^.OJ,. 

Starch  is  widely  distributed  throughout  the  vegetable  kingdom, 
occurring  in  grains,  fruits,  and  tubers.  It  occurs  in  granular  form,  the 
microscopical  appearance  being  typical  for  each  individual  starch. 
The  granules,  which  differ  in  size  according  to  the  source,  are  composed 
of  alternating  concentric  rings  of  granulose  and  cellulose.  Ordinary 
starch  is  insoluble  in  cold  water,  but  if  boiled  with  water  the  cell  walls 
are  ruptured  and  starch  paste  results.  In  general  starch  gives  a  blue 
color  with  iodine. 

Starch  is  acted  upon  by  amylases,  e.  g.,  salivary  amylase  {ptyalin) 
and  pancreatic  amylase  (amylopsin) ,  with  the  formation  of  soluble  starch, 
erythro-dextrin,  achroo-dextrins,  maltose,  iso-maltose  and  dextrose  (see 
Salivary  Digestion,  page  6i).  Maltose  is  the  principal  end-product  of 
this  enzyme  action.  Upon  boiling  a  starch  solution  with  a  dilute  mineral 
acid  a  series  of  similar  bodies  is  formed,  but  under  these  conditions 
dextrose  is  the  principal  end-product. 

Experiments  on  Starch. 

1.  Preparation  of  Potato  Starch. — ^Pare  a  raw  potato,  comminute 
it  upon  a  fine  grater,  mix  with  water,  and  "whip  up"  the  pulped  material 
^dgorously  before  straining  it  through  cheese  cloth  or  gauze  to  remove 
the  coarse  particles.  The  starch  rapidly  settles  to  the  bottom  and  can  be 
washed  by  repeated  decantation.  Allow  the  compact  mass  of  starch  to 
drain  thoroughly  and  spread  it  out  on  a  watch  glass  to  dry  in  the  air. 
If  so  desired  this  preparation  may  be  used  in  the  experiments  which  follow. 

2.  Microscopical  Examination. — Examine  microscopically  the  gran- 
ules of  the  various  starches  submitted  and  compare  them  with  those 
shown  in  Figs.  8-18,  page  49.  The  suspension  of  the  granules  in  a  drop 
of  water  will  facilitate  the  microscopical  examination. 

3.  Solubility. — Try  the  solubility  of  one  form  of  starch  in  each  of 
the  ordinary  solvents  (see  page  27).  If  uncertain  regarding  the  solubility 
in  any  reagent,  filter  and  test  the  filtrate  with  iodine  solution  as  given 


CARBOHYDRATES. 


49 


Fig.  8. — Potato. 


Fig.  g. — Bean. 


Fig.   io. — .'\rrowroot. 


Fig.  II. — Rye. 


Fig.  12. — Barley. 


Fig.  13. — Oat. 


Fig.  14. — Buckwheat. 


Fig.  15. — Maize. 


Fig    16. — KicE. 


Fig.  17.— Pea.  Fig.  18.— Wheat. 

Starch  Granules  krom  \'arious  Sources.     (Leffmai.n  and  Beam ) 


so  PHYSIOLOGICAL    CHEMISTRY. 

under  5  below.     The  production  of  a  blue  color  would  indicate  that  the 
starch  had  been  dissolved  by  the  solvent. 

4.  Iodine  Test. — ^Place  a  few  granules  of  starch  in  one  of  the  depres- 
sions of  a  porcelain  test-tablet  and  treat  with  a  drop  of  a  dilute  solution  of 
iodine  in  potassium  iodide.  The  granules  are  colored  blue  due  to  the 
formation  of  so-called  iodide  of  starch.  The  cellulose  of  the  granule  is 
not  stained  as  may  be  seen  by  examining  microscopically. 

5.  Iodine  Test  on  Starch  Paste/ — Repeat  the  iodine  test  using  the 
starch  paste.  Place  2-3  c.c.  of  starch  paste"  in  a  test-tube,  add  a  drop 
of  the  dilute  iodine  solution  and  observe  the  production  of  a  blue  color. 
Heat  the  tube  and  note  the  disappearance  of  the  color.  It  reappears  on 
cooling. 

In  similar  tests  note  the  influence  of  alcohol  and  of  alkali  upon  the 
so-called  iodide  of  starch. 

The  composition  of  the  iodide  of  starch  is  not  definitely  known. 

6.  Fehling's  Test. — On  starch  paste  (see  page  32). 

7.  Hydrolysis  of  Starch. — ^Place  about  25  c.c.  of  starch  paste  in  a 
small  beaker,  add  10  drops  of  concentrated  HCl,  and  boil.  By  means  of  a 
small  pipette,  at  the  end  of  each  minute,  remove  a  drop  of  the  solution  to 
the  test-tablet  and  make  the  regular  iodine  test.  As  the  testing  proceeds 
the  blue  color  should  gradually  fade  and  finally  disappear.  At  this  point, 
after  cooling  and  neutralizing  with  solid  KOH,  Fehling's  test  (see  page  32) 
should  give  a  positive  result  due  to  the  formation  of  a  reducing  sugar 
from  the  starch.  Make  the  phenylhydrazine  test  upon  some  of  the 
hydrolyzed  starch.     What  sugar  has  been  formed  ? 

8.  Influence  of  Tannic  Acid. — Add  an  excess  of  tannic  acid  solution 
to  a  small  amount  of  starch  paste  in  a  test-tube.  The  liquid  will  become 
strongly  opaque  and  ordinarily  a  yellowish-white  precipitate  is  produced. 
Compare  this  result  with  the  result  of  the  similar  experiment  on  dextrin 
(page  53). 

9.  Diffusibility  of  Starch  Paste. — Test  the  diffusibility  of  starch 
paste  through  animal  membrane,  parchment  paper  or  collodion,  making 
a  dialyzer  like  one  of  the  models  shown  in  Fig.  2,  page  30. 

mULIN,   (CeH,,0,),. 

Inulin  is  a  polysaccharide  which  may  be  obtained  as  a  white,  odorless, 
tasteless  powder  from  the  tubers  of  the  artichoke,  elecampane,  or  dahlia. 

'  Preparation  of  Si  arch  Paste. — Grind  2  grams  of  starch  powder  in  a  mortar  with  a  small 
amount  of  cold  water.  Bring  200  c.c.  of  water  to  the  boiling-point  and  add  the  starch  mixture 
from  the  mortar  with  continuous  stirring.  Bring  again  to  the  boiling-point  and  allow  it  to 
cool.  This  makes  an  approximate  1  per  cent  starch  paste  which  is  a  very  satisfactory  strength 
for  general  use. 

'  for  this  particular  test  a  starch  paste  of  very  satisfactory  strength  may  be  made  by 
mixing  i  c.c.  of  a  i  per  cent  starch  paste  with  100  c.c.  of  water. 


CARBOHYDRATES.  51 

It  has  also  been  prepared  from  the  roots  of  chicory,  dandelion,  and  ];ur- 
dock.  It  is  very  slightly  soluble  in  cold  water  and  quite  easily  soluble  in 
hot  water.  In  cold  alcohol  of  60  per  cent  or  over  it  is  practically  insoluble. 
Inulin  gives  a  negative  reaction  with  iodine  solution.  The  "yellow" 
color  reaction  with  iodine  mentioned  in  many  books  is  doubtless  nurely 
the  normal  color  of  the  iodine  solution.  It  is  very  difficult  to  prepare  inu- 
lin which  does  not  reduce  Fehling's  solution  slightly.  This  reducing 
power  may  be  due  to  an  impurity.  Practically  all  commercial  prepara- 
tions of  inulin  possess  considerable  reducing  power. 

Inulin  is  laevo-rotatory  and  upon  hydrolysis  by  acids  or  by  the  enzyme 
inulase  it  yields  the  monosaccharide  Itevulose  which  readily  reduces 
Fehling's  solution.  The  ordinary  amylolytic  enzymes  occurring  in  the 
animal  body  do  not  digest  inulin.  A  small  part  of  the  ingested  inulin 
may  be  hydrolized  by  the  acid  gastric  juice,  but  Lewis ^  has  recently 
shown  that  "  the  value  of  inulin  as  a  significant  source  of  energy  in  human 
dietaries  must  be  questioned." 


Experiments  on  Inulin. 

1.  Solubility. — Try  the  solubility  of  inulin  powder  in  each  of  the 
ordinary  solvents.  If  uncertain  regarding  the  solubility  in  any  reagent, 
filter  and  neutralize  the  filtrate  if  it  is  alkaline  in  reaction.  Add  a  drop  of 
concentrated  hydrochloric  acid  to  the  filtrate  and  boil  it  for  one  minute. 
Render  the  solution  neutral  or  slightly  alkaline  with  solid  potassium 
hydroxide  and  try  Fehling's  test.  What  is  the  significance  of  a  positive 
Fehling's  test  in  this  connection  ? 

2.  Iodine  Test.^ — (a)  Place  2-3  c.c.  of  the  inulin  solution  in  a  test- 
tube  and  add  a  drop  of  dilute  iodine  solution.     What  do  you  observe  ? 

(b)  Place  a  small  amount  of  inulin  powder  in  one  of  the  depressions 
of  a  test-tablet  and  add  a  drop  of  dilute  iodine  solution.  Is  the  effect  any 
different  from  that  observed  above  ? 

3.  Molisch's  Reaction. — Repeat  this  test  according  to  directions 
given  under  Dextrose,  2,  page  27. 

4.  Fehling's  Test. — Make  this  test  on  the  inulin  solution  according 
to  the  instructions  given  under  Dextrose,  page  32.  Is  there  any 
reduction  ?^ 

5.  Hydrolysis  of  Inulin. — Place  5  c.c.  of  inulin  solution  in  a  test-tube, 
add  a  drop  of  concentrated  hydrochloric  acid  and  boil  it  for  one  minute. 
Now  cool  the  solution,  neutralize  it  with  concentrated  KOH  and  test  the 

'  Lewis:  Journal  American  Medical  Ass'n.,  58,  1176,  1912. 
-  See  the  discussion  of  the  properties  of  inulin,  above. 


52  PHYSIOLOGICAL    CHEMISTRY. 

reducing  action  of  i  c.c.  of  the  solution  upon  i  c.c.  of  diluted  (i  14)  Feh- 
ling's  solution.     Explain  the  result.  ^ 

GLYCOGEN,   {C,U,,0,),. 

(For  discussion  and  experiments  see  Muscular  Tissue,  Chapter  XV.) 

LICHENIN,   (CgH^^OJ,. 

Lichenin  may  be  obtained  from  Cetraria  islandica  (Iceland  moss).  It 
forms  a  diflBicultly  soluble  jelly  in  cold  water  and  an  opalescent  solution  in 
hot  water.  It  is  optically  inactive  and  gives  no  color  with  iodine.  Upon 
hydrolysis  with  dilute  mineral  acids  lichenin  yields  dextrins  and  dextrose. 
It  is  said  to  be  most  nearly  related  chemically  to  starch.  Saliva,  pan- 
creatic juice,  malt  diastase  and  gastric  juice  have  no  noticeable  action  on 
lichenin. 

DEXTRIN,   (CeH^^OJ,. 

The  dextrins  are  the  bodies  formed  midway  in  the  stages  of  the 
hydrolysis  of  starch  by  weak  acids  or  an  enzyme.  They  are  amorphous 
bodies  which  are  easily  soluble  in  water,  acids,  and  alkalis,  but  are  insol- 
uble in  alcohol  or  ether.  Dextrins  are  dextro-rotatory  and  are  not  fer- 
mentable by  yeast. 

The  dextrins  may  be  hydrolyzed  by  dilute  acids  to  form  dextrose. 
With  iodine  one  form  of  dextrin  (erythro-dextrin)  gives  a  red  color.  Their 
power  to  reduce  Fehling's  solution  is  questioned. 

Experiments  on  Dextrin. 

1.  Solubility. — Test  the  solubility  of  pulverized  dextrin  in  the 
ordinary  solvents  (see  page  27). 

2.  Iodine  Test. — ^Place  a  drop  of  dextrin  solution  in  one  of  the 
depressions  of  the  test-tablet  and  add  a  drop  of  a  dilute  solution  of  iodine 
in  potassium  iodide.  A  red  color  results  due  to  the  formation  of  the  red 
iodide  of  dextrin.  If  the  reaction  is  not  sufficiently  pronounced  make  a 
stronger  solution  from  pulverized  dextrin  and  repeat  the  test.  The 
solution  should  be  slightly  acid  to  secure  the  best  results. 

Make  proper  tests  to  show  that  the  red  iodide  of  dextrin  is  influenced  by 
heat,  alkali,  and  alcohol  in  a  similar  manner  to  the  blue  iodide  of  starch 
(see  page  50). 

'  If  the  inulin  solution  gave  a  positive  Fehling  test  in  the  last  experiment  it  will  be 
necessary  to  check  the  hydrolysis  experiment  as  follows:  To  5  c.c.  of  the  inulin  solution  in  a 
test-tube  add  one  drop  of  concentrated  hydrochloric  acid,  neutralize  with  concentrated 
KOH  solution  and  test  the  reducing  action  of  i  c.c.  of  the  resulting  solution  upon  i  c.c. 
of  diluted  (i  :4)  Fehling's  solution.  This  will  show  the  normal  reducing  power  of  the  inulin 
solution.  In  case  the  inulin  was  hydrolyzed,  the  Fehling's  test  in  the  hydrolysis  experiment 
should  show  a  more  pronounced  reduction  than  that  observed  in  the  check  experiment. 


CARBOHYDRATES.  53 

3.  Fehling's  Test. — Sec  if  the  dextrin  solution  will  reduce  Fehling's 
solution. 

4.  Hydrolysis  of  Dextrin. — Take  25  c.c.  of  dextrin  solution  in  a 
small  beaker,  add  5  drops  of  dilute  hydrochloric  acid,  and  boil.  By 
means  of  a  small  pipette,  at  the  end  of  each  minute,  remove  a  drop  of 
the  solution  to  one  of  the  depressions  of  the  test-tablet  and  make  the 
iodine  test.  The  power  of  the  solution  to  produce  a  color  with  iodine 
should  rapidly  disappear.  When  a  negative  reaction  is  obtained  cool 
the  solution  and  neutralize  it  with  concentrated  potassium  hydroxide- 
Try  Fehling's  test  (see  page  32).  This  reaction  is  now  strongly  positive, 
due  to  the  formation  of  a  reducing  sugar.  Determine  the  nature  of  the 
sugar  by  means  of  the  phenylhydrazine  test  (see  pages  28  and  29). 

5.  Influence  of  Tannic  Acid. — Add  an  excess  of  tannic  acid  solution 
to  a  small  amount  of  dextrin  solution  in  a  test-tube.  No  precipitate 
forms.  This  result  differs  from  the  result  of  the  similar  experiment  upon 
starch  (see  Starch,  8,  page  50). 

6.  Diffusibility  of  Dextrin. — (See  Starch,  9,  page  50.) 

7.  Precipitation  by  Alcohol. — To  about  50  c.c.  of  95  per  cent 
alcohol  in  a  small  beaker  add  about  10  c.c.  of  a  concentrated  dextrin 
solution.  Dextrin  is  thrown  out  of  solution  as  a  gummy  white  precipitate. 
Compare  the  result  with  that  obtained  under  Dextrose,  5,  page  50. 

CELLULOSE,  (C^H^^OJ, 

This  complex  polysaccharide  forms  a  large  portion  of  the  cell  wall  of 
plants.  It  is  extremely  insoluble  and  its  molecule  is  much  more  complex 
than  the  starch  molecule.  The  best  quality  of  filter  paper  and  the  ordi- 
nary absorbent  cotton  are  good  types  of  cellulose. 

At  one  time  there  was  but  a  single  known  solvent  for  cellulose.  Recent 
investigation  has,  however,  revealed  a  long  list  of  cellulose  solvents.  (See 
Experiment  7.) 

Cellulose  is  not  hydrolyzed  by  boiling  with  dilute  mineral  acids.  It 
may  be  hydrolyzed,  however,  by  treating  with  concentrated  sulphuric 
acid  then  subsequently  diluting  the  solution  with  water  and  boiling. 

There  is  some  difference  of  opinion  as  to  the  exact  extent  to  which 
cellulose  is  utilized  in  the  animal  organism.  It  is  no  doubt,  more  effi- 
ciently utilized  by  herbivora  than  by  carnivora  or  by  man.  It  is  claimed 
that  about  25  per  cent  may  be  utilized  by  herbivora,  less  than  5  per  cent  by 
dogs  whereas  the  quantity  utilized  by  man  is  "too  small  for  it  to  play  a 
r6le  of  importance  in  the  diet  of  a  normal  individual."*  In  neither  man 
nor  the  lower  animals  has  there  been  demonstrated  any  formation  of 

'  Swartz:  Transactions  of  the  Connecticut  Academy  of  Arts  and  Sciences,  i6,  247,  191 1. 


54  PHYSIOLOGICAL   CHEMISTRY. 

sugar  or  glycogen  from  cellulose.  ^  It  is  probable  that  the  cellulose  which 
disappears  from  the  intestine  is  transformed  for  .the  most  part  into  fatty 
acids. ~ 

Experiments  on  Cellulose. 

1.  Solubility. — Test  the  solubility  of  cellulose  in  the  ordinary  solvents 
(see  page  27). 

2.  Iodine  Test. — Add  a  drop  of  dilute  iodine  solution  to  a  few  shreds 
of  cotton  on  a  test-tablet.  Cellulose  differs  from  starch  and  dextrin  in 
giving  no  color  with  iodine. 

3.  Formation  of  Amyloid.^ — Add  10  c.c.  of  dilute  and  5  c.c.  of 
concentrated  H2SO4  to  some  absorbent  cotton  in  a  test-tube.  When 
entirely  dissolved  (without  heating)  pour  one-half  of  the  solution  into 
another  test-tube,  cool  it  and  dilute  with  water.  Amyloid  forms  as 
a  gummy  precipitate  and  gives  a  brown  or  blue  coloration  with  iodine. 

After  allowing  the  second  portion  of  the  acid  solution  of  cotton  to  stand 
about  10  minutes,  dilute  it  with  water  in  a  small  beaker  and  boil  for  15-30 
minutes.  Now  cool,  neutralize  with  solid  KOH  and  test  with  Fehling's 
solution.  Dextrose  has  been  formed  from  the  cellulose  by  the  action  of 
the  acid. 

4.  Schweitzer's  Solubility  Test. — ^Place  a  little  absorbent  cotton  in  a 
test-tube,  add  Schweitzer's  reagent,^  and  stir  the  cellulose  with  a  glass 
rod.  When  completely  dissolved  acidify  the  solution  with  acetic  acid. 
An  amorphous  precipitate  of  cellulose  is  produced. 

5.  Cross  and  Bevan's  Solubility  Test.^ — ^Place  a  little  absorbent 
cotton  in  a  test-tube,  add  Cross  and  Bevan's  reagent,"  and  stir  the  cellulose 
with  a  glass  rod.  When  solution  is  complete  reprecipitate  the  cellulose 
with  95  per  cent  alcohol. 

6.  Iodine-Zinc  Chloride  Reaction. — Place  a  little  absorbent  cotton 
or  quantitative  filter  paper  in  a  test-tube  and  treat  it  with  the  iodine-zinc 
chloride  reagent.^     A  blue  color  forms  on  standing.     Amyloid  has  been 

'  Lusk:  American  Journal  of  Physiulogy,  27,  467,  1911;  also  Hoffmann,  Inaugural  dis- 
sertation, Halle-Wittenberg,  igio. 

^Tappeiner:  ZeUschrift  fiir  Biologie,  24,  105,  1888. 

^  This  bofly  derives  its  name  from  amylum  (starch)  and  is  not  to  be  confounded  with 
amyloid,  the  glycoprotein. 

*  Schweitzer's  reagent  is  made  by  adding  potassium  hydroxide  to  a  solution  of  copper 
sulphate  which  contains  some  ammonium  chloride.  A  precipitate  of  cupric  hydroxide 
forms  and  this  is  filtered  off,  washed,  and  3  grams  of  the  moist  cupric  hydroxide  brought 
into  solution  in  a  liter  of  20  per  cent  ammonium  hydroxide. 

^  Cross  and  Bevan:  Chemical  News,  63,  p.  66. 

®  Cross  and  Bevan's  reagent  may  be  prepared  by  combining  two  parts  of  concentrated 
hydrochloric  acid  and  one  part  of  zinc  chloride,  by  weight. 

'  The  iodine-zinc  chloride  reagent  as  suggested  by  Nowopokrowsky  (Beihefte  Bolan. 
Centr.,  28,  90,  1912)  may  be  made  by  dissolving  20  grams  ZnCl_,  in  8.5  c.c.  water  and  when 
cool  introducing  the  iodine  solution  (3  grams  KI-I- 1.5  gram  I  in  60  c.c.  water)  drop  by  drop 
until  iodine  begins  to  precipitate. 


CARBOHYDRATES.  55 

formed  from  the  cellulose  through  the  action  of  the  ZnCl,  and  the  iodine 
solution  has  stained  the  amyloid  blue. 

7.  New  Cellulose  Solvents. — It  has  recently  been  demonstrated  by 
Deming^  that  there  are  many  excellent  solvents  for  cellulose  (filter  paper). 
For  example,  the  concentrated  aqueous  solutions  of  certain  salts  such  as 
antimony  trichloride,  stannous  chloride  and  zinc  bromide.  In  hydro- 
chloric acid  solution  the  solvent  action  of  the  abo\'e  salts  is  increased. 
The  following'salts  are  also  good  solvents  in  hydrochloric  acid  solution: 
mercuric  chloride,  bismuth  chloride,  antimony  pentachloride,  tin  tetrachloride 
and  titanium  tetrachloride.  In  the  case  of  the  last-mentioned  salt  the 
swollen,  transparent  character  of  the  cellulose  libers  preliminary  to  solution 
can  be  seen  very  nicely. 

Try  selected  solvents  suggested  by  the  instructor. 

HEMICELLULOSES. 

The  hemicelluloses  differ  from  cellulose  in  that  they  may  be  hydrolysed 
upon  boiling  with  dilute  mineral  acids.  They  differ  from  other  poly- 
saccharides in  not  being  readily  digested  by  amylases.  Hemicellulose 
may  yield  pentosans,  or  hexosans  upon  hydrolysis. 

Pentosans. — ^Pentosans  yield  pentoses  upon  hydrolysis.  So  far  as  is 
known  they  do  not  occur  in  the  animal  kingdom.  They  have,  however, 
a  very  wide  distribution  in  the  vegetable  kingdom,  being  present  in  leaves, 
roots,  seeds  and  stems  of  all  forms  of  plants,  many  times  in  intimate 
association  or  even  chemical  combination  with  galactans.  In  herbivora, 
pentosans  are  40-80  per  cc^nt  utilized.'  The  few  tests  on  record  as  to  the 
pentosan  utilization  by  man^  indicate  that  80-95  P^r  cent  disappear  from 
the  intestine.  According  to  Cramer,*  bacteria  are  efficient  hemicellulose 
transformers.  It  has  not  yet  been  demonstrated  that  pentosans  form 
glycogen  in  man,  and  for  this  reason  they  must  be  considered  as  playing  an 
unimportant  part  in  human  nutrition.  Gum  arable  an  important  pento- 
san may  be  hydrolyzed  by  concentrated  hydrochloric  acid  if  boiled  for  a 
short  time.     The  pentose  arabinose  results  from  such  hydrolysis. 

Galactans. — In  common  with  the  pentosans  the  galactans  have  a  very 
wide  distribution  in  the  vegetable  kingdom.  The  pure  galactans  yield 
galactose  upon  hydrolysis.  One  of  the  most  important  members  of  the 
galactan  group  is  agar-agar,  a  product  prepared  from  certain  types  of 
Asiatic  sea-weed.  This  galactan  is  about  50  per  cent  utilizable  by 
herbivora^  and  8-27  per  cent  utilizable  by  man."     Agar  ingestion  has 

I  Deming:  Journal  American  Chemical  Society,  t,t„  1515,  iqii. 

-Swartz:  Transactions  of  the  Connecticut  Academy  of  Arts  and  Sciences,  16,  247,  1911. 

'  Konig  and  Reinhardt:  Zeit.  f.  Uutersuchung  der  Xalirungs  u.  Centissmitlel,  5,  110,1902. 

*  Cramer:  Inaug.  Diss.,  Halle,  1910. 

*  Lohrisch:  Zeit.f.  exper.  Path.  u.  Phartn.,  5,  478,  1908. 

*  Saiki:  /our.  Biol.  Cheni.,  2,  251,  1906. 


56  PHYSIOLOGICAL   CHEMISTRY. 

been  shown  to  be  a  very  efficient  therapeutic  aid  in  cases  of  chronic 
constipation.^'  ^  This  is  particularly  true  when  the  constipation  is  due 
to  the  formation  of  dry,  hard,  fecal  masses  (scybala),  a  type  of  fecal  forma- 
tion which  frequently  follows  the  ingestion  of  a  diet  which  is  very  thoroughly 
digested  and  absorbed.  The  agar,  because  of  its  relative  indigestibility 
and  its  property  of  absorbing  water  yields  a  bulky  fecal  mass  which  is 
sufficiently  soft  to  permit  of  easy  evacuation.  Agar  has  been  used  with 
good  results  in  the  treatment  of  constipation  in  children.^  The  function 
of  agar  is  not  limited  to  its  use  in  connection  with  constipation,  it  may 
serve  in  other  capacities  as  an  aid  to  intestinal  therapeutics.^ 

Experiments  on  a  Pentosan. 

1.  Solubility. — Test  the  solubility  of  gum  arable  in  the  ordinary 
solvents  (see  page  27). 

2.  Iodine  Test. — Add  a  drop  of  dilute  iodine  solution  to  a  little  gum 
arable  on  a  test-tablet.  It  resembles  cellulose  in  giving  no  color  with 
iodine. 

3.  Hydrolysis  of  Gum  Arabic. — Introduce  a  little  gum  arable  into  a 
test-tube,  add  5-10  c.c.  of  strong  hydrochloric  acid  (cone.  HCl  and  water 
1:1)  and  heat  to  boiling  for  5-10  minutes.  Cool,  neutralize  with 
potassium  hydroxide  and  test  by  the  Fehling  or  some  other  reduction  test. 
A  positive  reaction  should  be  obtained  indicating  that  the  gum  arable 
has  been  hydrolyzed  by  the  acid  with  the  production  of  a  reducing  sub- 
stance.    What  is  this  reducing  substance  ?     How  would  you  identify  it  ? 

Experiments  on  a  Galactan. 

1.  Solubility. — Test  the  solubility  of  agar-agar  in  the  ordinary  sol- 
vents (see  page  27).  Observe  its  marked  property  of  imbibing  water 
(see  page  255). 

2.  Iodine  Test. — Add  a  drop  of  dilute  iodine  solution  to  a  little 
agar-agar  on  a  test- tablet.  It  resembles  cellulose  in  giving  no  color 
with  iodine. 

3.  Hydrolysis  of  Agar-agar. — Introduce  a  few  pieces  of  agar-agar 
into  a  test-tube,  add  5-10  c.c.  of  strong  hydrochloric  acid  (cone.  HCl 
and  water  I  :i)  and  heat  to  boiling  for  5-10  minutes.  Cool,  neutralize 
with  potassium  hydroxide  and  test  by  the  Fehling  or  some  other  re- 
duction test.     A  positive  reaction  should  be  obtained  indicating  that 

'  Mendel:  Zentralblat,  f.  d.  ^esammle  Phys.  u.  Path,  des  Sloffw.,  No.  17,  i,  1908. 

^Schmidt:  Munch,  med.  Woch.,  52,  1970,  1905. 

'  Morse:  Journal  American  Medical  Ass'?i.,  55,  934,  1910. 

*  Einhorn:  Berl.  klin.  Woch.,  49,  113,  1912. 


CARBOHYDRATES. 


57 


the  agar-agar  has  been  hydrolyzed  by  the  acid  with  the  production  of  a 
reducing  substance.  What  is  this  reducing  substance?  How  would 
you  identify  it? 

REVIEW     OF     CARBOHYDRATES. 

In  order  to  facihtate  the  student's  ^e^'ie^v  of  the  carhobydrates,  the 
preparation  of  a  chart  similar  to  the  appended  model  is  recommended. 


MODEL  CHART  FOR  REVIEW  PURPOSES. 

Carbo- 
hydrate. 

Dextrose. 

1 
"o 

Iodine  Test. 
Moore's  Test. 

Trommer's  Test. 
Fehling's  Test. 
Boettger's  Test. 

m 

'u 
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Barfoed's  Test. 

Seliwanoff's 
Reaction. 

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Starch. 

Inulin. 

(Glycogen. 

Dextrin. 

— 

— 

— 



Cellulose. 

Gum  Arabic 

— 

— 

— 



— 

Agar-Agar 

The  signs  +  and  —  may  be  conveniently  used  to  indicate  positive 
and  negative  reaction.  Only  those  carbohydrates  which  are  of  greatest 
importance  from  the  standpoint  of  physiological  chemistry  have  been 
included  in  the  chart. 

"Unknown"  Solutions  of  Carbohydrates. 

At  this  point  the  student  will  be  given  several  "unknow^n"  solutions, 
each  solution  containing  one  or  more  of  the  carbohydrates  studied. 
He  will  be  required  to  detect,  by  means  of  the  tests  on  the  preceding 
pages,  each  carbohydrate  constituent  of  the  several  "unknown"  solutions 
and  hand  in,  to  the  instructor,  a  written  report  of  his  findings,  on  slips 
furnished  by  the  laboratory. 

The  scheme  given  on  page  58  may  be  of  use  in  this  connection. 


58 


PHYSIOLOGICAL   CHEMISTRY. 


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CHAPTER  III. 
SALIVARY  DIGESTION. 

The  saliva  i?  secreted  by  three  pairs  of  glands,  the  submaxillary, 
sublingual,  and  parotid,  reinforced  by  numerous  small  glands  called 
buccal  glands.  The  saliva  secreted  by  each  pair  of  glands  possesses 
certain  definite  characteristics  peculiar  to  itself.  For  instance,  in  man 
the  parotid  glands  ordinarily  secrete  a  thin,  watery  fluid,  the  submaxillary 
glands  secrete  a  somewhat  thicker  fluid  containing  mucin,  while  the 
product  of  the  sublingual  glands  has  a  more  mucilaginous  character. 
The  saliva  as  collected  from  the  mouth  is  the  combined  product  of  all  the 
glands  mentioned.  The  fact  that  there  are  pronounced  variations  in  the 
composition  of  dift'erent  fractions  of  saliva  secreted  by  the  same  normal 
individual  on  a  uniform  diet  has  recently  been  emphasized  by  Lothrop 
and  Gies.^ 

The  saliva  may  be  induced  to  flow  by  many  forms  of  stimuli,  such  as 
chemical,  mechanical,  electrical,  thermal,  and  psychical,  the  nature  and 
amount  of  the  secretion  depending,  to  a  limited  degree,  upon  the  par- 
ticular class  of  stimuli  employed  as  well  as  upon  the  character  of  the 
indindual  stimulus.  For  example,  in  experiments  upon  dogs  it  has  been 
found  that  the  mechanical  stimulus  afforded  by  dropping  several  pebbles 
into  the  animal's  mouth  caused  the  flow  of  but  one  or  two  drops  of  saliva, 
whereas  the  mechanical  stimulus  afforded  by  sand  thrown  into  the  mouth 
induced  a  copious  flow  of  a  thin  watery  fluid.  Again,  when  ice- water  or 
snow  was  placed  in  the  animal's  mouth  no  saliva  was  seen,  while  an  acid 
or  anything  possessing  a  bitter  taste,  which  the  dog  wished  to  reject, 
caused  a  free  flow  of  the  thin  saliva.  On  the  other  hand,  when  articles 
of  food  were  placed  in  the  dog's  mouth  the  animal  secreted  a  thicker 
saliva  having  a  higher  mucin  content — a  fluid  which  would  lubricate  the 
food  and  assist  in  the  passage  of  the  bolus  through  the  oesophagus.  It 
was  further  found  that  by  simply  drawing  the  attention  of  the  animal  to 
any  of  the  substances  named  above,  results  were  obtained  similar  to  those 
secured  when  the  substances  were  actually  placed  in  the  animal's  mouth. 
For  example,  when  a  pretense  was  made  of  throwing  sand  into  the  dog's 
mouth,  a  watery  saliva  was  secreted,  whereas  food  under  the  same  con- 
ditions excited  a  thicker  and  more  slimy  secretion.  The  exhibition  of  dry 
food,  in  which  the  dog  had  no  particular  interest  (dry  bread)  caused  the 

'  Lothrop  and  Gies:  Journal  of  the  Allied  {Dental)  Societies,  6,  65,  1911. 

59 


6o  PHYSIOLOGICAL   CHEMISTRY, 

secretion  of  a  large  amount  of  watery  saliva,  while  the  presentation  of 
moist  food,  which  was  eagerly  desired  by  the  animal,  called  forth  a  much 
smaller  secretion,  slimy  in  character.  These  experiments  show  it  to  be 
rather  difficult  to  differentiate  between  the  influence  of  physiological  and 
psychical  stimuli. 

The  amount  of  saliva  secreted  by  an  adult  in  24  hours  has  been  vari- 
ously placed,  as  the  result  of  experiment  and  observation,  between  1000 
and  1500  c.c,  the  exact  amount  depending,  among  other  conditions,  upon 
the  character  of  the  food. 

The  saliva  of  adults  ordinarily  has  a  weak,  alkaline  reaction  to  litmus, 
but  becomes  acid,  in  some  instances,  2-3  hours  after  a  meal  or  during  fast- 
ing. The  saliva  of  the  newborn  is  generally  neutral  to  litmus,  whereas  that 
of  infants,  especially  those  breast-fed,  is  generally  acid.  ^  The  alkalinity  of 
saliva  is  due  principally  to  di-sodium  hydrogen  phosphate  (NajHPOJ 
and  its  average  alkalinity  may  be  said  to  be  equivalent  to  0.08-0.1  per 
cent  sodium  carbonate.  The  saliva  is  the  most  dilute  of  all  the  digestive 
secretions,  having  an  average  specific  gravity  of  1.005  ^^^  containing  only 
o.  5  per  cent  of  solid  matter.  Among  the  solids  are  found  albumin,  globulin, 
mucin,  urea,  the  enzymes  salivary  amylase  (ptyalin),  maltase,  and  peptide 
splitting  enzymes;  phosphates,  and  other  inorganic  constituents.  Potas- 
sium thiocyanate,  KSCN,  is  also  generally  present  in  the  saliva.  It  has 
been  claimed  that  this  substance  is  present  in  greatest  amount  in  the  saliva 
of  habitual  smokers.  The  significance  of  thiocyanate  in  the  saliva  is  not 
known;  it  probably  comes  from  the  ingested  thiocyanates  and  from  the 
breaking  down  of  protein  material.  The  attempts  to  show  some  relation- 
ship between  tooth  decay  and  the  thiocyanate  content  of  the  saliva 
secreted  into  the  mouth  cavity  have  met  with  failure.  The  most  recent 
experiments^  indicate  a  virtual  absence  of  such  relationship. 

The  so-called  tartar  formation  on  the  teeth  is  composed  almost 
entirely  of  calcium  phosphate  with  some  calcium  carbonate,  mucin, 
epithelial  cells,  and  organic  debris  derived  from  the  food.  The  calcium 
salts  are  held  in  solution  as  acid  salts,  and  are  probably  precipitated  by 
the  ammonia  of  the  breath.  The  various  organic  substances  just  men- 
tioned are  carried  down  in  the  precipitation  of  the  calcium  salts. 

The  suggestion  has  been  made  that  mucin  is  the  salivary  constituent 
"  which  is  particularly  influential  in  the  development  of  local  conditions 
favoring  the  onset  of  dental  decay. "^ 

The  principal  enzyme  of  the  saliva  is  known  as  salivary  amylase  or 
ptyalin.  This  is  an  amylolytic  enzyme  (see  page  4),  so  called  because  it 
possesses  the  property  of  transforming  complex  carbohydrates  such  as 

'  Allaria:  Monalsschr.  fiir  Kinderheilkunde,  lo,  179,  191 1. 

^  Lothrop  and  Gies:  Journal  of  the  Allied  {Dental)  Societies,  6,  65,  1911. 

*Id.;  Ibid.,  5,  No.  4,  1910. 


SALIVARY    DIGESTION.  6l 

starch  and  dextrin  into  simpler  bodies.  The  action  of  salivary  amylase 
is  one  of  hydrolysis  and  through  this  action  a  series  of  simpler  bodies  are 
formed  from  the  complex  starch.  The  first  product  of  the  action  of  the 
ptyalin  of  the  saliva  upon  starch  paste  is  soluble  starch  (amidulin)  and  its 
formation  is  indicated  by  the  disappearance  of  the  opalescence  of  the 
starch  solution.  This  body  resembles  true  starch  in  giving  a  blue  color 
with  iodine.  Next  follows  the  formation,  in  succession,  of  a  series  of 
dextrins,  called  drylliro-dcxtrin,  a-achroo-dextrin,  ^-achroo-dextrin,  and 
y-achroo-dextrin,  the  erythro-dextrm  being  formed  directly  from  soluble 
starch  and  later  being  itself  transformed  into  a-achroo-dextrin  from  which 
in  turn  are  produced  ^-achroo-dextrin,  y-achroo-dextrin  and  perhaps  other 
dextrins.  Accompanying  each  dextrin  a  small  amount  of  iso-maltose  is 
formed,  the  quantity  of  iso-maltose  growing  gradually  larger  as  the  process 
of  transformation  progresses.  (Erythro-dextrin  gives  a  red  color  with 
iodine,  the  other  dextrins  give  no  color.)  The  next  stage  is  the  transforma- 
tion of  the  final  dextrin  into  f50-wa//05e  and  subsequently  the  transforma- 
tion of  the  iso-maltose  into  maltose,  the  latter  being  the  principal  end- 
product  of  the  salivary  digestion  of  starch.  At  this  point  a  small  amount 
of  dextrose  is  formed  from  the  maltose,  through  the  action  of  the  enzyme 
maltase. 

Salivary  amylase  acts  in  alkaline,  neutral,  or  combined  acid  solu- 
tions. It  will  act  in  the  presence  of  relatively  strong  combined  HCl  (see 
page  126),  whereas  a  trace  (0.003  P^i"  cent  to  0.006  per  cent)  of  ordinary 
free  hydrochloric  acid  will  not  only  prevent  the  action  but  will  destroy  the 
enzyme.  By  sufficiently  increasing  the  alkalinity  of  the  saliva  to  litmus, 
the  action  of  the  salivary  amylase  is  inhibited. 

It  has  been  claimed  by  Roger  ^  that  the  activation  of  human  saliva 
inactivated  by  the  action  of  heat  or  hydrochloric  acid  could  be  brought 
about  by  the  addition  of  traces  of  fresh  human  saliva.  Very  recent  at- 
tempts- to  verify  this  claim  have  met  with  failure. 

It  has  recently  been  shown  by  Cannon  that  salivary  digestion  may 
proceed  for  a  considerable  period  after  the  food  reaches  the  stomach, 
owing  to  the  slowness  with  which  the  contents  are  thoroughly  mixed  with 
the  acid  gastric  juice  and  the  consequent  tardy  destruction  of  the  enzyme. 
Food  in  the  pyloric  end  of  the  stomach  is  soon  mixed  with  the  gastric 
secretion,  but  food  in  the  cardiac  end  is  not  mixed  with  the  acid  gastric 
juice  for  a  considerable  period  of  time,  and  in  this  region  during  that  time 
salivary  digestion  may  proceed  undisturbed. 

It  has  very  recently  been  found  that  salivary  amylase  acts  more 
eflSciently  when  the  saliva  is  diluted.^     The  optimum  dilution  for  sodium 

'  Roger:  Rev.  Gen.  des.  ScL,  i8,  544,  IQ07. 
-  Bergeim  and  Hawk:  Unpublished  data. 
*Bergeim  and  Hawk:  Unpublished  data. 


62  PHYSIOLOGICAL    CHEMISTRY. 

chloride  solution  (0.3  per  cent)  was  found  to  be  about  four  volumes, 
whereas  that  for  tap  water ^  and  distilled  water  was  about  seven  volumes. 
These  findings  are  of  interest  in  connection  with  the  more  efficient  utili- 
zation of  ingested  carbohydrate  which  has  been  found  to  accompany  the 
drinking  of  large  volumes  of  water  at  meal  time.^  Lipase  also  acts  better  , 
in  dilution.^ 

It  has  further  been  demonstrated  very  recently  that  the  action  of  sali- 
vary amylase  is  inhibited  in  the  presence  of  softened  water.''  The 
inhibitory  factor  was  found  to  be  magnesium  hydroxide.^  Electrolytes 
have  an  important  influence  upon  the  action  of  amylases.  The  CI  ion 
has  a  pronounced  facilitating  action  (see  Pancreatic  Amylase). 

The  question  of  the  adaptation  of  the  salivary  secretion  to  diet  is  one 
which  has  received  considerable  attention  in  recent  years.  It  has  been 
claimed,  on  the  basis  of  experimental  evidence,^  that  the  continued  feeding 
of  a  carbohydrate  diet  causes  the  secretion  of  a  saliva  which  contains  a 
higher  concentration  of  salivary  amylase  and  one  which  is  therefore  able  to 
more  efficiently  digest  the  carbohydrate  fed.  On  the  other  hand  strong 
evidence^  has  been  submitted  that  the  amylase  content  of  the  saliva  is  not 
increased  through  the  continued  feeding  of  a  carbohydrate  diet.  The 
balance  of  evidence  is  however  opposed  to  adaptation.  In  general 
the  concensus  of  opinion  is  opposed  to  the  adaptation  of  digestive  secretions 
to  diet. 

Maltase,  sometimes  called  glucase,  is  the  second  enzyme  of  the  saliva. 
The  principal  function  of  maltase  is  the  splitting  of  maltose  into  dextrose. 
Besides  occurring  in  the  saliva  it  is  also  present  in  the  pancreatic  and 
intestinal  juices.  For  experimental  purposes  the  enzyme  is  ordinarily 
prepared  from  corn.  The  principles  of  the  "reversibility"  of  enzyme 
action  were  first  demonstrated  in  connection  with  maltase  by  Croft  Hill. 

The  presence  in  the  saliva  of  dipeptide-  and  tripeptide-splitting 
enzymes  has  recently  been  demonstrated.*  Leucyl-glycyl-alanine  was 
the  tripeptide  split  whereas  the  cleavage  of  several  dipeptids  was 
brought  about.  The  action  is  similar  to  that  of  intestinal  erepsin  (see 
Chapter  VIII). 

Microscopical  examination  of  the  saliva  reveals  salivary  corpuscles, 

'  University  of  Illinois  Water  Supply. 

^  Mattill  and  Hawk:  Jour.  Am.  Chem.  Soc,  33,  2019,  1911. 

^Bradley:    Jour.  Biol.  Chem.,  8,  251,  1910. 

*  Prepared  by  treating  top  water  with  one-sixth  its  volume  of  saturated  lime  water,  allowing 
to  stand  24  hours  and  filtering. 

'  Bergeim  and  Hawk:  Unpublished  data. 

"  Neilson  and  Terry:  American  Journal  of  Physiology,  15,  406,  1905;  Neilson  and  Lewis: 
Journal  of  Biological  Chemistry,  4,  501,  1908. 

^Mendel:  American  Journal  of  the  Medical  Sciences,  Oct.,  1909;  Mendel  and  Underbill: 
Journal  of  Biological  Chemistry,  3,  135,  1907.  Mendel,  Chapman  and  Blood:  Medical 
Record,  Aug.  27,  1910. 

*  Koelker:  Zeitschrift  fiir  physiol.  Chem.,  76,  27,  191 1. 


SALIVARY    DIGESTION.  63 

bacteria,  food  debris,  epithelial  cells,  mucus,  and  fungi.  In  certain 
pathological  conditions  of  the  mouth,  pus  cells,  and  blood  corpuscles  may 
be  found  in  the  saliva. 

Experiments  on  Saliva. 

A  satisfactory  method  of  obtaining  the  saliva  necessary  for  the  experi- 
ments which  follow  is  to  chew  a  small  piece  of  pure  paralBn  wax,  thus 
stimulating  the  flow  of  the  secretion,  which  may  be  collected  in  a  small 
beaker.  Filtered  saliva  is  to  be  used  in  every  experiment  except  for  the 
microscopical  examination. 

I.  Microscopical  Examination. — Examine  a  drop  of  unfiltered 
saliva  microscopically  and  compare  with  Fig.  19  below. 


Fig.  19. — Microscopical  Constituents  of  Saliva. 
a,  Epithelial  cells;  b,  salivary  corpuscles;  c    fat  drops;  d,  leucocytes;  e,  /  and  g.    bacteria; 

h,  i  and  k,  fission-fungi. 

2.  Reaction. — Test  the  reaction  to  litmus,  phenolphthalein  and 
Congo  red. 

3.  Specific  Gravity. — Partially  fill  a  urinometer  cylinder  with  saliva, 
introduce  the  urinometer,  and  observe  the  reading. 

4.  Test  for  Mucin. — To  a  small  amount  of  saliva  in  a  test-tube  add 
1-2  drops  of  dilute  acetic  acid.     Mucin  is  precipitated. 

5.  Biuret  Test.^ — Render  a  little  saliva  alkaline  with  an  equal  volume 
of  KOH  and  add  a  few  drops  of  a  very  dilute  (2-5  drops  in  a  test-tube  of 
water)  copper  sulphate  solution.  The  formation  of  a  purplish-violet 
color  is  due  to  mucin. 

6.  Millon's  Reaction.- — Add  a  few  drops  of  Millon's  reagent  to  a 
little  saliva.  A  light  yellow  precipitate  formed  by  the  mucin  gradually 
turns  red  upon  being  gently  heated. 

7.  Preparation  of  Mucin. — Pour  25  c.c.  of  saliva  into  100  c.c.  of 
95  per  cent  alcohol,  stirring  constantly.  Cover  the  vessel  and  allow  the 
precipitate  to  stand  at  least  12  hours.  Pour  off  the  supernatant  liquid, 
collect  the  precipitate  on  a  filter  and  wash  it,  in  turn,  with  alcohol  and 

'  The  significance  of  this  reaction  is  pointed  out  on  page  98. 
-  The  significance  of  this  reaction  is  pointed  out  on  page  97. 


64  PHYSIOLOGICAL    CHEMISTRY, 

ether.  Finally  dry  the  precipitate,  remove  it  from  the  paper  and  make 
the  following  tests  on  the  mucin:  (a)  Test  its  solubility  in  the  ordinary 
solvents  (see  page  27);  (b)  Millon's  reaction;  (c)  dissolve  a  small  amount 
in  KOH,  and  try  the  biuret  test  on  the  solution;  (d)  boil  the  remainder, 
with  10-25  c.c.  of  water  to  which  5  c.c.  of  dilute  HCl  has  been  added, 
until  the  solution  becomes  brownish.  Cool,  render  alkaline  with  solid 
KOH,  and  test  by  Fehling's  solution.  A  reduction  should  take  place. 
Mucin  is  what  is  known  as  a  conjugated  protein  or  glycoprotein  (see 
p.  94)  and  upon  boiling  wdth  the  acid  the  carbohydrate  group  in  the  mole- 
cule has  been  split  off  from  the  protein  portion  and  its  presence  is  indicated 
by  the  reduction  of  Fehling's  solution. 

8.  Inorganic  Matter. — Test  for  chlorides,  phosphates,  sulphates,  and 
calcium.  For  chlorides,  acidify  with  HNO3  and  add  AgXOg.  For 
phosphates,  acidify  with  HNO3,  ^^^^  ^^^  ^.dd  molybdic  solution.^  For 
sulphates,  acidify  with  HCl  and  add  BaCl,  and  warm.  For  calcium, 
acidify  with  acetic  acid,  CH3COOH,  and  add  ammonium  oxalate, 
(NHJX.O,. 

9.  Viscosity  Test. — Place  filter  papers  in  two  funnels,  and  to  each 
add  an  equal  quantity  of  starch  paste  (5  c.c).  Add  a  few  drops  of  saliva 
to  one  lot  of  paste  and  an  equivalent  amount  of  water  to  the  other.  Xote 
the  progress  of  filtration  in  each  case.  ^^Tiy  does  one  solution  filter  more 
rapidly  than  the  other  ? 

10.  Test  for  Nitrites.— Add  1-2  drops  of  dilute  HjSO^  to  a  little 
saliva  and  thoroughly  stir.  Now  add  a  few  drops  of  a  potassium  iodide 
solution  and  some  starch  paste.  Nitrous  acid  is  formed  which  liberates 
iodine,  causing  the  formation  of  the  blue  iodide  of  starch. 

11.  Thiocyanate  Tests. — (a)  Ferric  Chloride  Test. — To  a  little  saliva 
in  a  small  porcelain  crucible,  or  dish,  add  a  few  drops  of  dilute  ferric 
chloride  and  acidify  slightly  with  HCl.  Red  ferric  thiocyanate  forms. 
To  show  that  the  red  coloration  is  not  due  to  iron  phosphate  add  a  drop 
of  HgClj  when  colorless  mercuric  thiocyanate  forms. 

(6)  Solera's  Reaction. — This  test  depends  upon  the  liberation  of  iodine 
through  the  action  of  thiocyanate  upon  iodic  acid.  Moisten  a  strip  of 
starch  paste-iodic  acid  test  paper^  with  a  little  saliva.  If  thiocyanate  be 
present  the  test  paper  will  assume  a  blue  color,  due  to  the  liberation  of 
iodine  and  the  subsequent  formation  of  the  so-called  iodide  of  starch. 

*  Molybdic  solution  is  prepared  as  follows,  the  parts  being  by  weight: 
I  part  molybdic  acid. 

4  parts  ammonium  hydroxide  (sp.  gr.  0.96). 
15  parts  nitric  acid  (sp.  gr.  i.  2). 
^  This  test  paper  is  prepared  as  follows:  Saturate  a  good  quality  of  filter  paper  with  0.5 
per  cent  starch    paste  to  which  has  been  added  sufficient  iodic  acii  to  make  a  i  per  cent 
solution  of  iodic  acid  and  allow  the  paper  to  dry  in  the  air.     Cut  it  in  strips  of  suitable  size  and 
preserve  for  use. 


SALIVARY    DIGESTION  65 

12.  Digestion  of  Starch  Paste. — To  25  c.c.  of  starch  paste  in  a  small 
beaker,  add  5  drops  of  saliva  and  stir  thoroughly.  At  intervals  of  a  minute 
remove  a  drop  of  the  solution  to  one  of  the  depressions  in  a  test-tablet  and 
test  by  the  iodine  test.  If  the  blue  color  with  iodine  still  forms  after  5 
minutes,  add  another  5  drops  of  saliva.  The  opalescence  of  the  starch 
solution  should  soon  disappear,  indicating  the  formation  of  soluble  starch 
which  gives  a  blue  color  with  iodine.  This  body  should  soon  be  trans- 
formed into  erythro-dextrin  which  gives  a  red  color  with  iodine,  and  this  in 
turn  should  pass  into  achroo-dextrin  which  gives  no  color  with  iodine. 
This  is  called  the  achromic  point.  When  this  point  is  reached  test  by 
Fehling's  test  to  show  the  production  of  a  reducing  body.  A  positive 
Fehling's  test  may  be  obtained  while  the  solution  still  reacts  red  with 
iodine  inasmuch  as  some  iso-maltose  is  formed  from  the  soluble  starch 
coincidently  with  the  formation  of  the  erythro-dextrin.  How  long  did  it 
take  for  a  complete  transformation  of  the  starch  ? 

13.  Digestion  of  Dry  Starch. — In  a  test-tube  shake  up  a  small 
amount  of  dry  starch  with  a  little  water.  Add  a  few  drops  of  saliva,  mix 
well,  and  allow  to  stand.  After  10-20  minutes  filter  and  test  the  filtrate 
by  Fehling's  test.     What  is  the  result  and  why  ? 

14.  Digestion  of  Inulin. — To  5  c.c.  of  inulin  solution  in  a  test-tube 
add  ID  drops  of  saliva  and  place  the  tube  in  the  incubator  or  water-bath 
at  40°  C.  After  one-half  hour  test  the  solution  by  Fehling's  test.  ^  Is  any 
reducing  substance  present?  What  do  you  conclude  regarding  the 
salivary  digestion  of  inulin  ? 

15.  Influence  of  Temperature. — In  each  of  four  tubes  place  about 
5  c.c.  of  starch  paste.  Immerse  one  tube  in  cold  water  from  the  faucet, 
keep  a  second  at  room  temperature,  and  place  a  third  in  the  incubator  or 
the  water-bath  at  40°  C.  Now  add  to  the  contents  of  each  of  these  three 
tubes  two  drops  of  saliva  and  shake  well;  to  the  contents  of  the  fourth 
tube  add  two  drops  of  boiled  saliva.  Test  frequently  by  the  iodine  test, 
using  the  test-tablet,  and  note  in  which  tube  the  most  rapid  digestion 
occurs.     Explain  the  results. 

16.  Influence  of  Dilution."— Take  a  series  of  six  test-tubes  each 
containing  9  c.c.  of  water.  Add  i  c.c.  of  saliva  to  tube  i  and  shake 
thoroughly.  Remove  i  c.c.  of  the  solution  from  tube  i  to  tube  2  and 
after  mixing  thoroughly  remove  i  c.c.  from  tube  2  to  tube  3.  Continue 
in  this  manner  until  you  have  6  saliva  solutions  of  gradually  decreasing 
strength.     Now  add  starch  paste  in  equal  amounts  to  each  tube,  mix 

*  If  the  inulin  solution  gives  a  reduction  before  being  acted  upon  b)'  the  saliva  it  wrill  be 
necessary  to  determine  the  extent  of  the  original  reduction  by  means  of  a  "check"  test  (see 
page  52). 

^  The  technic  of  Wohlgemuth's  method  (see  page  i8)  may  be  employed  in  this  test  if 
so  desired. 


66  PHYSIOLOGICAL    CHEMISTRY. 

very  thoroughly,  and  place  in  the  incubator  or  water-bath  at  40°  C. 
After  10-20  minutes  test  by  both  the  iodine  and  Fehling's  tests.  In  how 
great  dilution  does  your  saliva  act  ? 

17.  Influence  of  Acids  and  Alkalis. — {a)  Influence  of  Free  Acid. — 
Prepare  a  series  of  six  tubes  in  each  of  which  is  placed  4  c.c.  of  one  of  the 
follo\nng  strengths  oifree  HCl:  0.2  per  cent,  o.i  per  cent,  0.05  per  cent, 
0.025  per  cent,  0.0125  per  cent  and  0.006  per  cent.  Now  add  2  c.c.  of 
starch  paste  to  each  tube  and  shake  them  thoroughly.  Complete  the 
solutions  by  adding  2  c.c.  of  saliva  to  each  and  repeat  the  shaking.  The 
total  acidity  of  this  series  would  be  as  follows:  o.i  per  cent,  0.05  per  cent, 
0.025  per  cent,  0.0125  per  cent,  0.006  per  cent  and  0.003  P^^  cent.  Place 
these  tubes  on  the  water-bath  at  40°  C.  for  10-20  minutes.  Divide  the 
contents  of  each  tube  into  two  parts,  testing  one  part  by  the  iodine  test 
and  testing  the  other,  after  neutralization,  by  Fehling's  test.  What  do 
you  find  ? 

(&)  Influence  of  Combined  Acid  {Protein  Salt). — Repeat  the  first  three 
experiments  of  the  above  series  using  combined  hydrochloric  acid  (see 
page  126)  instead  of  the/ree  acid.  How  does  the  action  of  the  combined 
acid  differ  from  that  of  \htfree  acid?  (For  a  discussion  of  combined  acid 
see  page  126.J 

(c)  Influence  of  Alkali. — Repeat  the  first  four  experiments  under  (a) 
replacing  the  HCl  by  2  per  cent,  i  per  cent,  0.5  per  cent  and  0.25  per  cent 
NajCOg.  Neutralize  the  alkalinity  before  trying  the  iodine  test  (see 
Starch,  5,  page  50). 

(d)  Nature  of  the  Action  of  Acid  and  Alkali. — ^Place  2  c.c.  of  saliva  and 
2  c.c.  of  0.2  per  cent  HCl  in  a  test-tube  and  leave  for  15  minutes.  Neu- 
tralize the  solution,  add  4  c.c.  of  starch  paste  and  place  the  tube  in  the 
incubator  or  water-bath  at  40°  C.  In  10  minutes  test  by  the  iodine  and 
Fehling's  tests  and  explain  the  result.  Repeat  the  experiment,  replacing 
the  0.2  per  cent  HCl  by  2  per  cent  Na2C03.  Whd.i  do  you  deduce  from 
these  two  experiments  ? 

18.  Influence  of  Metallic  Salts,  etc. — In  each  of  a  series  of  tubes 
place  4  c.c.  of  starch  paste  and  1/2  c.c.  of  one  of  the  solutions  named  below. 
Shake  well,  add  1/2  c.c.  of  saliva  to  each  tube,  thoroughly  mix,  and  place 
in  the  incubator  or  water-bath  at  40°  C.  for  10-20  minutes.  Show  the 
progress  of  digestion  by  means  of  the  iodine  and  Fehling  tests.  Use  the 
following  chemicals:  Metallic  salts,  10  per  cent  lead  acetate,  2  per 
cent  copper  sulphate,  5  per  cent  ferric  chloride,  8  per  cent  mercuric 
chloride;  Neutral  salts,  10  per  cent  sodium  chloride,  10  per  cent  mag- 
nesium sulphate,  3  per  cent  barium  chloride,  10  per  cent  Rochelle  salt. 
Also  try  the  influence  of  2  per  cent  carbolic  acid,  95  per  cent  alcohol,  and 
ether  and  chloroform.     What  are  your  conclusions  ? 


SALIVARY   DIGESTION  67 

19.  Excretion  of  Potassium  Iodide. — Ingest  a  small  dose  of  potas- 
sium iodide  (0.2  gram)  contained  in  a  gelatin  capsule,  quickly  rinse  out 
the  mouth  with  water,  and  then  test  the  saliva  at  once  for  iodine.  This 
test  should  be  negative.  Make  additional  tests  for  iodine  at  2-minute 
intervals.  The  test  for  iodine  is  made  as  follows:  Take  i  c.c.  of  NaNOz 
and  I  c.c.  of  dilute  H.SO/  in  a  test-tube,  add  a  little  saliva  directly 
from  the  mouth,  and  a  small  amount  of  starch  paste.  The  formation  of 
a  blue  color  signifies  that  the  potassium  iodide  is  being  excreted  through 
the  salivary  glands.  Note  the  length  of  time  elapsing  between  the  inges- 
tion of  the  potassium  iodide  and  the  appearance  of  the  first  traces  of  the 
substance  in  the  saliva.  If  convenient,  the  urine  may  also  be  tested. 
The  chemical  reactions  taking  place  in  this  experiment  are  indicated  in 
the  following  equations : 

(a)  2NaN03  +  H^SO,— 2HNO3  +  Na^SO,. 

(b)  2KI  +  H2SO,  — 2HI  +  K2SO,. 

(c)  2HN02-h   2HI— I2  +  2H2O-I-2NO. 

20.  Qualitative  Analysis  of  the  Products  of  Salivary  Digestion. — 

To  25  c.c.  of  the  products  of  salivary  digestion  (saved  from  Experiment 
12  or  furnished  by  the  instructor),  add  100  c.c.  of  95  per  cent  alcohol. 
Allow  to  stand  until  the  white  precipitate  has  settled.  Filter,  evaporate 
the  filtrate  to  dryness,  dissolve  the  residue  in  5-10  c.c.  of  water  and  try 
Fehling's  test  (p.  32)  and  the  phenylhydrazine  reaction  (see  Dextrose,  3, 
page  28).  On  the  dextrin  precipitate  try  the  iodine  test  (page  50).  Also 
hydrolyze  the  dextrin  as  given  under  Dextrin,  4,  page  53. 

'  Instead  of  this  mixture  a  few  drops  of  HNO3  possessing  a  yellowish  or  brownish  color 
due  to  the  presence  of  HNO2  may  be  employed. 


CH.\PTER  IV. 
PROTEINS:^    THEIR  DECOMPOSITION  AND  SYNTHESIS. 

The  proteins  are  a  class  of  substances,  which  in  the  light  of  our  present 
knowledge,  consist,  in  the  main,  of  combinations  of  a-amino-acids  or 
their  derivatives.  These  protein  substances  form  the  chief  constituents 
of  many  of  the  fluids  of  the  body,  constitute  the  organic  basis  of  animal 
tissue,  and  at  the  same  time  occupy  a  decidedly  preeminent  position  among 
our  organic  food-stuffs.  They  are  absolutely  necessary  to  the  uses  of  the 
animal  organism  for  the  continuance  of  life  and  they  cannot  be  satis- 
factorily replaced  in  the  diet  of  such  an  organism  by  any  other  dietary 
constituent  either  organic  or  inorganic.  Such  an  organism  may  exist 
mthout  protein  food  for  a  period  of  time,  the  length  of  the  period  varying 
according  to  the  specific  organism  and  the  nature  of  the  substitution 
offered  for  the  protein  portion  of  the  diet.  Such  a  period  is,  however, 
distinctly  one  of  existence  rather  than  one  of  normal  life  and  one  which  is 
consequently  not  accompanied  by  such  a  full  and  free  exercise  of  the 
various  functions  of  the  organism  as  would  be  possible  upon  an  evenly 
balanced  ration,  i.  e.,  one  containing  the  requisite  amount  of  protein  food. 
These  protein  substances  are,  furthermore,  essential  constituents  of  all 
living  cells  and  therefore  without  them  vegetable  life  as  well  as  animal  life 
is  impossible. 

The  proteins,  which  constitute  such  an  important  group  of  substances, 
differ  from  the  carbohydrates  and  fats  very  decidedly  in  elementary  com- 
position. In  addition  to  containing  carbon,  hydrogen,  and  oxygen,  which 
are  present  in  fats  and  carbohydrates,  the  proteins  invariably  contain 
nitrogen  in  their  molecule  and  generally  sulphur  also.  Proteins  have  also 
been  described  which  contain  phosphorus,  iron,  copper,  iodine,  manganese, 
and  zinc.  The  percentage  composition  of  the  more  important  members 
of  the  group  of  protein  substances  would  fall  within  the  following  limits : 
0  =  50-55  per  cent,  H  =  6-7.3  P^^  cent,  0=19-24  per  cent,  N=  15-19  per 
cent,  5=0.3-2.5  per  cent,  P=o.4-o.8  per  cent  when  present.  When  iron, 
copper,  iodine,  manganese,  or  zinc  are  present  in  the  protein  molecule  they 
are  practically  without  exception  present  only  in  traces  and  with  the 
exception  of  iodine  are  probably  not  constituents  of  the  protein  molecule.^ 

*  The  term  proteid  has  been  very  widely  used  by  English-speaking  scientists  to  signify 
the  class  of  substances  we  have  called   proteins. 

*  Some  investigators  regard  these  elements  as  contaminations,  or  constituents  of  some 
non-protein  substance  combined  with  the  protein. 

68 


PROTEINS.  69 

Of  all  the  various  elements  of  the  protein  molecule,  nitrogen  is  by  far 
the  most  important.  The  human  body  needs  nitrogen  for  the  continua- 
tion of  life,  but  it  cannot  use  the  nitrogen  of  the  air  or  that  in  various  other 
combinations  as  we  lind  it  in  nitrates,  nitrites,  etc.  However,  in  the  pro- 
tein molecule  the  nitrogen  is  present  in  a  form  which  is  utilizable  by  the 
body.  The  nitrogen  in  the  protein  molecule  occurs  in  at  least  four 
different  forms  as  follows: 

I.  Monamino  acid  nitrogen. 
II.  Diamino  acid  nitrogen  or  banc  nitrogen. 

III.  Amide  nitrogen. 

IV.  A  guanidine  residue. 

The  actual  structure  of  the  protein  molecule  is  stilf  unknown,  and  we 
have  as  yet  no  means  by  which  its  molecular  weight  can  be  even  approxi- 
mately established.  The  many  attempts  which  have  been  made  to  deter- 
mine this  have  led  to  very  different  results,  some  of  which  are  given  in  the 
following  table: 

Globin  =15000  —  16086 

Oxyhaemoglobin     =  14800  — 15000— 16655  — 16730 

Of  these  figures,  those  given  for  oxyhaemoglobin  deserve  the  most 
consideration,  for  these  are  based  on  the  atomic  ratios  of  the  sulphur 
and  iron  contained  in  this  substance.  The  simplest  formula  that  can  be 
calculated  from  analyses  of  oxyhaemoglobin,  namely,  CesgHugiNjoySj- 
FeOjjo,  serves  to  show  the  great  complexity  of  this  substance. 

The  decomposition^  of  protein  substances  may  be  brought  about  by 
oxidation  or  hydrolysis,  but  inasmuch  as  the  hydrolytic  procedure  has 
been  productive  of  the  more  satisfactory  results,  that  type  of  decomposition 
procedure  alone  is  used  at  present.  This  hydrolysis  of  the  protein  mole- 
cule may  be  accomplished  by  acids,  alkalis,  or  superheated  steam,  and  in 
digestion  by  the  action  of  the  proteolytic  enzymes.  The  character  of  the 
decomposition  products  varies  according  to  the  method  utilized  in  tearing 
the  molecule  apart'.  Bearing  this  in  mind,  we  may  say  that  the  decom- 
position products  of  proteins  include  proteoses,  peptones,  peptides,  carbon 
dioxide,  ammonia,  hydrogen  sulphide,  and  amino  acids.  These  amino  acids 
constitute  a  long  list  of  important  substances  which  contain  nuclei  belong- 
ing either  to  the  aliphatic,  carbocyclic,  or  heterocyclic  series.  The  list 
includes  glycocoll,  alanine,  serine,  phenylalanine,  tyrosine,  cystine,  trypto- 
phane, histidine,  valine,  arginine,  leucine,  isoleucine,  lysine,  asparlic  acid, 
glutamic  acid,  proline,  oxyproline,  and  diaminotrihydroxydodecanoic  acid. 

'The  terms  "degradation,"  "dissociation,"  and  "cleavage,"  are  often  used  in  this 
connection. 


70  PHYSIOLOGICAL   CHEMISTRY. 

Of  these  amino  acids,  tyrosine  and  phenylalanine  contain  carbocyclic 
nuclei:  histidine,  proline,  and  tryptophane  contain  heterocyclic  nuclei: 
and  the  remaining  members  of  the  list,  as  given,  contain  aliphatic  nuclei. 
The  amino  acids  are  preeminently  the  most  important  class  of  protein 
decomposition  products.  These  amino  acids  are  all  a-amino  acids,  and, 
^A^ith  the  exception  of  glycocoll,  are  all  optically  active.  Furthermore, 
they  are  amphoteric  substances  and  consequently  are  able  to  form  salts 
with  both  bases  and  acids.  These  properties  are  inherent  in  the  NHj  and 
COOH  groups  of  the  amino  acids. 

The  decomposition  products  of  protein  may  be  grouped  as  primary 
and  secondary  decomposition  products.  By  primary  products  are  meant 
those  which  exist  as  radicals  within  the  protein  molecule  and  which  are 
liberated,  upon  cleavage  of  this  molecule,  with  their  carbon  chains  intact 
and  the  position  of  their  nitrogen  unaltered.  The  secondary  products  are 
those  which  result  from  the  disintegration  of  the  primary  cleaA^age  prod- 
ucts. No  matter  what  method  is  used  to  decompose  a  given  protein 
molecule,  the  primary  products  are  largely  the  same  under  all  conditions.^ 

In  the  process  of  hydrolysis  the  protein  molecule  is  gradually  broken 
down  and  less  complicated  aggregates  than  the  original  molecule  are 
formed,  which  are  known  as  proteoses,  peptones,  and  peptides,  and  which 
still  possess  true  protein  characteristics.  Further  hydrolysis  causes  the 
ultimate  transformation  of  these  substances,  of  a  protein  nature,  into  the 
amino  acids  of  known  chemical  structure.  In  this  decomposition  the 
protein  molecule  is  not  broken  down  in  a  regular  manner  into  1/2,  1/4, 
1/8  portions  and  the  amino  acids  formed  in  a  group  at  the  termination  of 
the  hydrolysis.  On  the  contrary,  certain  amino  acids  are  formed  very 
early  in  the  process,  in  fact  while  the  main  hydrolytic  action  has  pro- 
ceeded no  further  than  the  proteose  stage.  Gradually  the  complexity 
of  the  protein  portion  undergoing  decomposition  is  simplified  by  the  split- 
ting off  of  the  amino  acids  and  finally  it  is  so  far  decomposed  through  pre- 
vious cleavages  that  it  yields  only  amino  acids  at  the  succeeding  cleavage. 
In  short,  the  general  plan  of  the  hydrolysis  of  the  protein  molecule  is 
similar  to  the  hydrolysis  of  starch.  In  the  case  of  starch  there  is 
formed  a  series  of  dextrins  of  gradually  decreasing  complexity  and  coin- 
cidently  with  the  formation  of  each  dextrin  a  small  amount  of  sugar  is 
split  off  and  finally  nothing  but  sugar  remains.  In  the  case  of  protein 
hydrolysis  there  is  a  series  of  proteins  of  gradually  decreasing  complexity 
produced  and  coincidently  with  the  formation  of  each  new  protein  sub- 
stance amino  acids  are  split  off  and  finally  the  sole  products  remaining 
are  amino  acids. 

'  Alkaline  hydrolysis  yields  urea  and  ornithine  which  result  from  arginine,  the  product  of 
acid  hydrolysis. 


PROTEINS.  71 

Inasmuch  as  diversity  in  the  method  of  decomposing  a  given  protein 
does  not  result  in  an  cc[ually  diversified  line  of  decomposition  products, 
but,  on  the  other  hand,  yields  products  which  are  quite  comparable  in 
character,  it  may  be  argued  that  there  are  probably  well-defined  lines  of 
cleavage  in  the  indi\'idual  protein  molecule  and  that  no  matter  what  the 
force  brought  to  bear  to  tear  such  a  molecule  apart,  the  disintegration, 
when  it  comes,  will  yield  in  every  case  certain  definite  fragments.  These 
fragments  may  be  called  the  "building  stones"  of  the  protein  molecule,  a 
term  used  by  some  of  the  German  investigators.  Take,  for  example,  the 
decomposition  of  protein  which  may  be  brought  about  through  the  action 
of  the  enzyme  trypsin  of  the  pancreatic  juice.  When  this  enzyme  is  allowed 
to  act  upon  a  given  protein,  the  latter  is  disintegrated  in  a  series  of  definite 
cleavages,  resulting  in  the  formation  of  proteoses,  peptones,  and  peptides 
in  regular  order,  the  peptides  being  the  last  of  the  decomposition  products 
which  possess  protein  characteristics.  They  are  all  built  up  from 
amino  acids  and  are  therefore  closely  related  to  these  acids  on  the  one 
side  and  to  peptones  on  the  other.  We  have  di-,  tri-,  tetra-,  penta-,  deca-, 
and  poly-peptides  which  are  named  according  to  the  number  of  amino 
acids  included  in  the  peptide  molecule.  Following  the  peptides  there  are 
a  diverse  assortment  of  monamino  and  diamino  acids  which  constitute  the 
final  products  of  the  protein  decomposition.  These  acids  are  devoid  of 
any  protein  characteristics  and  are  therefore  decidedly  different  from  the 
original  substance  from  which  they  were  derived.  From  a  protein  of  huge 
molecular  weight,  a  typical  colloid,  perhaps  but  slightly  soluble,  and 
entirely  non-diffusible,  we  have  passed  by  way  of  proteoses,  peptones,  and 
peptides  to  a  class  of  simpler  crystalline  substances  which  are,  for  the  most 
part,  readily  soluble  and  diffusible. 

These  amino  acids  after  their  production  in  the  process  of  digestion, 
as  just  indicated,  are  synthesized  within  the  organism  to  form  protein 
material  which  goes  to  build  up  the  tissues  of  the  body.  It  is  thus  seen 
that  the  amino  acids  are  of  prime  importance  in  the  animal  economy.  It 
was  formerly  believed  that  these  essential  factors  in  metabolism  and 
nutrition  could  not  be  produced  within  the  animal  organism  from  their  ele- 
ments, but  were  only  yielded  upon  the  hydrolysis  of  ingested  protein  of 
animal  or  vegetable  origin.  Recent  experiments,  however,  by  Abderhol- 
den  and  by  Grafe  and  Schlapfcr  and  others  indicate  that  the  nitrogen 
of  food  protein  may  in  part  be  replaced  by  ammonium  salts.  Experi- 
ments by  Osborne  and  others  also  indicate  amino  acid  synthesis  by 
animals. 

There  are  formed,  by  life  processes  in  both  the  animal  and  the 
vegetable  kingdom  certain  transformation  products  of  amino  acids. 
Our  knowledge  regarding  these  has  been  advanced  principally  through  the 


72 


PHYSIOLOGICAL    CHEMISTRY. 


efforts  of  Kutscher  and  his  colleagues.  This  class  of  substances  has 
been  given  the  name  aporrhegmas.^  Among  the  aporrhegmas  are 
included  acids  and  bases  formed  in  putrefaction  as  well  as  a  number  of 
similar  compounds  which  have  not  been  isolated  from  putrefaction  mix- 
tures but  are  formed  normally  in  the  plant  or  animal  body.  (For  further 
discussion  of  aporrhegmas,  see  chapter  on  Putrefaction.) 

Important  data  regarding  the  decomposition  products  of  the  protein 
molecule  are  given  in  the  tables  which  follow. 


COMPARISON  OF  THE  DECOINIPOSITION  PRODUCTS  OF  PROTAMINES,  AND 

OTHER  PROTEINS. 


I  Protamines. - 

(Per  cent  of  total  nitrogen  of 
amino  acid). 


Other  Proteins. 
(Per  cent  of  amino  acids  in 
proteins.) 


4-      ....      +      +      .... 

2  .  00 

3.6 

i-S 

0.8       4.2 

9.79 

Valine 

+           +           4-        T.65 

3-34 

6.2 

7.2 

I.O    ' 

1.88 

+     

6.62 

14-5 

9.4 

2.1    Izg.o 

19-55 

Proline 

....     3  .8' 1+43 

13.22 

4.1 

6.7 

5-2    1    2.3 

9.04 

Phenylalanine 

2-3  5 

31 

3-2 

0.4    :   4-2 

6.55 

Aspartic  acid 

0.58 

4-5 

1-4 

0.56    4.4 

I. 71 

Glutamic  acid  .  .  .  . 

43.66 

18.74 

II  .0 

i.88i    1.7 

26.17 

+325 

O.I3 

0.33 

o.S 

0.4   1   0.6 

I  .  20 

2 . 1 

4-5 

0!    1.3 

3.55 

Arginine 

.  .  .  .  88.8  67.7  63.  5     8.7  28.0  88.0  89.2 

3.16 

14.2 

4.84 

7.62[    5.4 

i.SS 

0 

1-7 

S.9S 

2-75    4-3 

Histidine 

II. 8 

0.61 

2 .  2 

2.50 

1 
0.4011 .0 

0.82 

Tryptophane 

+        + 

I  .0 

4- 

I  -5 

0       4- 

0 

_ 

1 .00    0.065      ° 

0.3 

J 

Oxyproline ....  i ....  i .  .      '          1                                   ' 

2.0      0.23    6.4 

I  .0 

f 

Diaminotrihydroxydo- 1 .  .  .  . 

0.7S 

1 

? 

decanoic  acid.                  [          ;          , 

....  1 

5.22   ] 

2.3 

1. 61 

3.64 

1 

'  Ackermann  and  Kutscher:  Zeit.  physiol.  Chent.,  69,  263,  1910;  Ackermann:  Ibid.,  273:  Engeland 
and  Kutscher:  Ibid.,  282. 

^  Kossel:  Zeit.  physiol.  Chem.,  44,  347,  1905. 

'  Osborne  and  Guest:  Jour.  Biol.  Chem.,  9,  425,  1911. 

<  Abderhalden,  Kcsseland  others. 

'  Abderhalden,  Fischer,  Momer  and  others. 

•Fischer,  Levene  and  Aders:  Zeit.  physiol.  Chem.,  35,  70,  1902;  also  Levene  and  Beatty:  Ibid,  49, 
252,  1906. 

'  Abderhalden :  Zei/.  physiol.  Chem.,  37^  484,  1903. 

'  Osborne  and  Liddle,  Am.  Jour.  Physiol.,  26,  295,  1910. 

*  This  unique  and  important  protein  has  probably  been  more  carefully  analyzed  than  any  other. 


FK(JTi;iN.S. 


73 


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t/i   rt 

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tx. 

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p-<. 


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74 


PHYSIOLOGICAL   CHEMISTRY. 


o  o 

*»  s 

C  'in 
O  O 

.^  a. 


PL, 


O 


u 


--3        T3 
ra         p! 


pq 


C  o5 

-a  :3 


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o 


n 


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pq 


u 


pj 


CX'XJ 


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3  3 


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PROTEINS.  75 

When  we  examine  the  formulas  of  the  principal  members  of  the 
crystalline  end-products  of  protein  decomposition  we  note  that  they  are 
invariably  acids,  as  has  already  been  mentioned,  and  contain  an  NH2 
group  in  the  a  position.  This  relation  of  the  NH,  group  to  the  acid  radi- 
cal is  constant,  no  matter  what  other  groups  or  radicals  are  present.  We 
may  have  straight  chains  as  in  alanine  and  glutamic  acid,  the  benzene  ring 
as  in  phenylalanine,  or  we  may  have  sulphurized  bodies  as  in  cystine  and 
still  the  formula  is  always  of  the  same  type,  i.  e., 

NH, 

I 
R— CH— COOH 

It  is  seen  that  this  characteristic  grouping  in  the  amino  acid  provides 
each  one  of  these  ultimate  fragments  of  the  protein  molecule  with  both  a 
strong  acid  and  a  strong  basic  group.  For  this  reason  it  is  theoretically 
possible  for  a  large  number  of  these  amino  acids  to  combine  and  the  result- 
ing combinations  may  be  very  great  in  number,  since  there  is  such  a  varied 
assortment  of  the  acids.  The  protein  molecule,  which  is  of  such  mam- 
moth proportions,  is  probably  constructed  on  a  foundation  of  this  sort. 
Of  late  much  valuable  data  have  been  collected  regarding  the  synthetic 
production  of  protein  substances,  the  leaders  in  this  line  of  investigation 
being  Fischer  and  Abderhalden.  After  ha\dng  gathered  a  mass  of  data 
regarding  the  final  products  of  the  protein  decomposition  and  demonstrat- 
ing that  amino  acids  were  the  ultimate  results  of  the  various  forms  of 
decomposition,  these  investigators,  and  notably  Fischer,  set  about  in  an 
effort  to  form,  from  these  amino  acids,  by  synthetic  means,  substances 
which  should  possess  protein  characteristics.  The  simplest  of  these 
bodies  formed  in  this  way  was  synthesized  from  two  molecules  of  glyco- 
coll  with  the  liberation  of  water,  thus: 


CH,-(NH,)-CO    OH  H   HN-CH^-COOH. 

The  body  thus  formed  is  a  dipeptide,  called  glycyl- glycine.  In  an  analo- 
gous manner  may  be  produced  leucyl-leucine,  through  the  synthesis  of 
two  molecules  of  leucine  or  leucyl-alanyl- glycine  through  the  union  of  one 
molecule  of  leucine,  one  of  alanine,  and  one  of  glycocoll.  By  this  pro- 
cedure Fischer  and  his  pupils  have  been  able  to  make  a  large  number  of 
peptides  containing  varied  numbers  of  amino-acid  radicals,  the  name 
polypeptides  being  given  to  the  whole  group  of  synthetic  substances  thus 
formed.  The  most  complex  poplypeptide  yet  produced  is  one  containing 
fifteen  glycocoll  and  three  leucine  residues. 

Notwithstanding  the  fact  that  most  synthetic  polypeptides  are  pro- 
duced through  a  union  of  amino  acids  bv  means  of  their  imide  bonds,  it 


76  PHYSIOLOGICAL   CHEMISTRY. 

must  not  be  imagined  that  the  protein  molecule  is  constructed  from  amino 
acids  linked  together  in  straight  chains  in  a  manner  analogous  to  the 
formation  of  simple  peptides,  such  as  glycyl-glycine.  The  molecular 
structure  of  the  proteins  is  much  too  complex  to  be  explained  upon  any 
such  simple  formation  as  that.  There  must  be  a  variety  of  linkings,  since 
there  is  a  varied  assortment  of  decomposition  products  of  totally  different 
structure. 

Many  of  these  synthetic  bodies  respond  to  the  biuret  test,  are  precipi- 
tated by  phosphotungstic  acid,  and  behave,  in  other  ways,  as  to  leave  no 
doubt  as  to  their  protein  characteristics.  For  instance,  a  number  of 
amino  acids  each  possessing  a  sweet  taste  have  been  synthesized  in  such  a 
manner  as  to  yield  a  polypeptide  of  hitter  taste,  a  well-known  characteristic 
of  peptones.  From  the  fact  that  the  polypeptides  formed  in  the  manner 
indicated  have  free  acidic  and  basic  radicals  we  gather  the  explanation  of 
the  amphoteric  character  of  true  proteins. 

For  the  benefit  of  those  especially  interested  in  such  matters  a  photo- 
graph of  the  Fischer  apparatus  (Fig.  23,  page  80)  used  in  the  fractional 
distillation,  in  vacuo,  of  the  esters  of  the  decomposition  products  of  the 
proteins,  as  well  as  micro-photographs  and  drawings  of  preparations  of 
several  of  these  decomposition  products  (Figs.  20  to  32,  pages  77  to  89) 
are  introduced.  For  the  preparations  and  the  photograph  of  the  appa- 
ratus the  author  is  indebted  to  Dr.  T.  B.  Osborne,  of  New  Haven,  Conn., 
who  has  made  many  important  observations  upon  the  hydrolysis  of 
proteins.  The  reproduction  of  the  crystalline  form  of  some  of  the  more 
recent  of  the  products  may  be  of  interest  to  those  viewing  the  field  of 
physiological  chemistry  from  other  than  the  student's  aspect. 

An  extended  discussion  of  the  various  decomposition  products  being 
out  of  place  in  a  book  of  this  character,  we  will  simply  make  a  few  general 
statements  in  connection  with  the  primary  decomposition  products. 


DISCUSSION  OF  THE  PRODUCTS. 

Ammonia,  NH3. — Ammonia  is  an  important  decomposition  product 
of  all  proteins  and  probably  arises  from  an  amide  group  combined 
with  a  carboxyl  group  of  some  of  the  amino  acids.  It  is  possible  that  the 
dibasic  acids,  aspartic  and  glutamic,  furnish  most  of  these  carboxyl 
groups.  This  is  indicated  by  the  more  or  less  close  relationship  which 
exists  between  the  amount  of  ammonia  and  that  of  the  dibasic  acids  which 
the  several  proteins  yield  upon  decomposition.  The  elimination  of  the 
ammonia  from  proteins  under  the  action  of  acids  and  alkalis  is  very 
similar  to  that  from  amides  like  asparagine. 


PROTEINS. 


77 


Glycocoll,  C2H5NO2. — Glycocoll,  or  amino  acetic  acid,  is  the  simplest 
of  the  amino  acids  and  has  the  following  formula: 

NH, 

I 
H— C— COOH. 

1 
H 

Glycocoll,  as  the*  formula  shows,  contains  no  asymmetric  carbon  atom, 
and  is  the  only  amino  acid  yielded  by  protein  decomposition  which  is 
optically  inactive.  Glycocoll  and  leucine  were  among  the  first  decom- 
position products  of  proteins  to  be  discovered.  Upon  administering  benzoic 
acid  to  animals  the  output  of  hippuric  acid  in  the  urine  is  greatly  increased, 


Fig.  20. — Glycocoll  Ester  Hydrochloride. 

thus  showing  a  synthesis  of  benzoic  acid  and  glycocoll  in  the  organism 
(see  page  168,  Chapter  IX).  Glycocoll,  ingested  in  small  amount,  is 
excreted  in  the  urine  as  urea,  whereas  if  administered  in  excess  it  appears 
in  part  unchanged  in  the  urine.  It  is  usually  separated  from  the  mixture 
of  protein  decomposition  products  as  the  hydrochloride  of  the  ester.  The 
crystalline  form  of  this  compound  is  shown  in  Fig.  20. 

Alanine,  CjH^NOg. — Alanine  is  a-amino-propionic  acid,  and  as  such 
it  may  be  represented  structurally  as  follows : 

H     NH2 

I       i 
H— C— C— COOH. 

1       I 
H    H 

Obtained  from  protein  substances,  alanine  is  dextro-rotatory,  is  very 
soluble  in  water,  and  possesses  a  sweet  taste.     Tyrosine,  phenylalanine, 


78 


PHYSIOLOGICAL    CHEMISTRY. 


cystine,  and  serine  are  derivatives  of  alanine.  This  amino  acid  has  been 
obtained  from  nearly  all  proteins  examined.  Its  absence  from  those 
proteins  from  which  it  has  not  been  obtained  has  not  been  proven.  Most 
proteins  yield  relatively  small  amounts  of  alanine. 

Serine,  C3H7NO3. — Serine  is  a-amino-^-hydroxy-propionic  acid  and 
possesses  the  following  structural  formula: 

OH  NH, 

I        I      " 
H  — C  — C— COOH 

H      H 

Serine  obtained  from  proteins  is  laevo-rotatory,  possesses  a  sweet  taste, 
and  is  quite  soluble  in  water.  Serine  is  not  obtained  in  quantity  from 
most  proteins,  but  is  yielded  abundantly  by  silk  glue.  Owing  to  the 
difficulty  of  separating  serine  it  has  not  been  found  in  a  '  number  of 
proteins  in  which  it  probably  occurs.  Serine_crystals  are  shown  in  Fig. 
2T,  below. 


Fig.  21. — Serine. 


Phenylalanine,    CgHjjNO,. — This    product    is    ^-phenyl- a-amino- 
propionic  acid,  and  may  be  represented  graphically  as  follows: 

H    NH, 

I       I     " 
C-C-COOH. 

I       I 
H    H 


The  Isevo-rotatory  form  is  obtained  from  proteins.  Phenylalanine  has 
been  obtained  from  all  the  proteins  examined  except  from  the  protamines 
and  some  of  the  albuminoids.     The  yield  of  this  body  from  the  decom- 


PROTEINS, 


79 
The 


position  of  proteins  is  frequently  greater  than  the  yield  of  tyrosine, 
crystalline  form  of  phenylalanine  is  shown  in  Fig.  22. 

Tyrosine,  CgHjjNOg. — Tyrosine,  one  of  the  first  discovered  end- 
products  of  protein  decomposition,  is  the  amino  acid,  p-ox y- .3 -phenyl- a- 
amino-propianic  acid.     It  has  the  following  formula: 

H    NH2 

I      I 
C-C-COOH. 


H    H 

OH 

The  tyrosine  which  results  from  protein  decomposition  is  usually  Icevo- 
rotatory.  Tyrosine  is  one  of  the  end-products  of  tryptic  digestion  and 
usually  separates  in  conspicuous  amount  early  in  the  process  of  digestion. 


Fig.  22. — Phexyl.\laxixe. 

It  does  not  occur,  however,  as  an  end-product  of  the  decomposition  of 
gelatin. 

Tyrosine  is  found  in  old  cheese,  and  derives  its  name  from  this  fact. 
It  crystallizes  in  tufts,  sheaves,  or  balls  of  fine  needles,  which  decompose 
at  295°  C.  and  are  sparingly  soluble  in  cold  (1-2454)  water,  but  much 
more  so  in  boiling  (1-154)  water.  Tyrosine  forms  soluble  salts  with 
alkalis,  ammonia,  or  mineral  acids,  and  is  soluble,  with  difficulty,  in 
acetic  acid.  It  responds  to  Millon's  reaction,  thus  showing  the  presence 
of  the  hydroxyphenyl  group,  but  gives  no  other  protein  test.  The  aro- 
matic groups  present  in  tyrosine,  phenylalanine,  and  tryptophane  cause 
proteins  to  yield  a  positive  xanthoproteic  reaction.  In  severe  cases  of 
typhoid  fever  and  smallpox,  in  acute  yellow  atrophy  of  the  liver,  and  in 


8o 


PHYSIOLOGICAL    CHEMISTRY. 


acute  phosphorus  poisoning,  tyrosine  has  been  found  in  the  urine.     Tyro- 
sine crystals  are  shown  in  Fig.  24,  page  81. 


Fig.  23. — Fischer  Apparatus. 

Reproduced  from  a  phoiograph  made  by  Prof.  E.  T.  Reichert,  of  the  University  of  Penn- 
sylvania.    The  negative  was  furnished  by  Dr.  T.  B.  Osborne,  of  New  Haven,  Conn. 

A,  Tank  into  which  freezing  mixture  is  pumped  and  from  whicla  it  flows  through  the 
condenser,  B;  C,  fiask  from  which  the  esters  are  distilled,  the  distillate  being  collected  in  D; 
E,  a  Dewar  flask  containing  liquid  air  serving  as  a  cooler  for  condensing  tube  F;  G  and  G', 
tubes  leading  to  the  Geryck  pump  by  which  the  vacuum  is  maintained;  /,  tube  leading  to  a 
McLeod  gauge  (not  shown  in  figure) ;  J,  a  bath  containing  freezing  mixture  in  which  the 
receiver  D  is  immersed;  K,  a  bath  of  water  during  the  first  part  of  the  distillation  and  of 
oil  during  the  last  part  of  the  process;  1-5,  stop  cocks  which  permit  the  cutting  out  of  different 
parts  of  the  apparatus  as  the  procedure  demands. 

Cystine,  CgHj204N2S2. — Friedmann  has  recently  shown  cystine  to 
be  a-diamino-[i-dithiolactyl  acid  and  to  possess  the  following  structural 
formula : 

CH^S-SCH, 

I  I 

CHNH,    CHNH, 


COOH      COOH 


PROTEINS.  8 1 


Cystine  is  the  principal  sulphur-containing  body  obtained  from  the 
decomposition  of  protein  substances.  It  is  obtained  in  greatest  amount 
as  a  decomposition  product  of  such  keratin-containing  tissues  as  horn, 


Fig.  24. — Tyrosine. 

hoof,  and  hair.  Cystine  occurs  in  small  amount  in  normal  urine  and  is 
greatly  increased  in  quantity  under  certain  pathological  conditions.  It 
crystallizes  in  thin,  colorless,  hexagonal  plates  which  are  shown  in  Fig.  25. 
Cystine  is  very  slightly  soluble  in  water  but  its  salts,  with  both  bases  and 
acids,  are  readily  soluble  in  water.     It  is  laevo-rotatory. 


Fig.  25. — Cystixe. 

It  was  formerly  claimed  that  cystine  occurred  in  two  forms,  i.  e., 
stone-cystine  and  protein-cystine  and  that  these  two  forms  are  distinct  in 
their  properties.     This  view  is  incorrect. 
6 


82  PHYSIOLOGICAL   CHEMISTRY. 

For  a  discussion  of  cystine  sediments  in  urine  see  Chapter  XX. 
Tryptophane,  CiiHj2N202. — Recently  Ellinger  and  Flamand  have 
shown  that  tryptophane  possesses  the  following  formula : 

/\ C-CH2-CH(NH,)-C00H 

CH 


NH 

It  is  therefore  indol-a-amino-propionic  acid.  Tryptophane  is  the 
mother-substance  of  indole,  skatole,  skatole  acetic  acid  and  skatole  carhoxylic 
acid,  all  of  which  are  formed  as  secondary  decomposition  products  of 
proteins.  Its  presence  in  protein  substances  may  be  shown  by  means  of 
the  Adamkiewicz  reaction  or  the  Hopkins-Cole  reaction  (see  page  98). 
It  may  be  detected  in  a  tryptic  digestion  mixture  through  its  property  of 
giving  a  violet  color-reaction  with  bromine  water.  Tryptophane  is 
yielded  by  nearly  all  proteins,  but  has  been  shown  to  be  entirely  absent  from 
zein,  the  prolamin  (alcohol-soluble  protein)  of  maize  and  also  from  gelatin. 

Upon  being  heated  to  285°  C.  tryptophane  decomposes  with  the  evolu- 
tion of  gas. 

Histidine,  CgHgNgO,. — Histidine  is  a-amino-^-imidazol-propionic 
acid  with  the  following  structural  formula : 

H    NH, 

I       I 
HC   -    C-C-C-COOH. 

I       I 
H    H 

HN.    ^N 
CH 

The  histidine  obtained  from  proteins  is  laevo-rotatory.  It  has  been 
obtained  from  all  the  proteins  thus  far  examined,  the  majority  of  them 
yielding  about  2.5  per  cent  of  the  amino  acid.  However,  about  11  per 
cent  was  obtained  by  Abderhalden  from  globin,  the  protein  constituent  of 
oxy haemoglobin  and  about  13  per  cent  by  Kossel  and  Kutscher  from  the 
protamine  sturine. 

Crystals  of  histidine  dichloride  are  shown  in  Fig.  26,  page  83. 

Knoop^s  Color  Reaction  for  Histidine. — To  an  aqueous  solution  of 
histidine  or  a  histidine  salt  in  a  test-tube  add  a  little  bromine  water.  A 
yellow  coloration  develops  in  the  cold  and  upon  further  addition  of  bro- 
mine water  becomes  permanent.  If  the  tube  be  heated,^  the  color  will 
disappear  and  will  shortly  be  replaced  by  a  faint  red  coloration  which 

•  The  same  reaction  will  take  place  in  the  cold  more  slowly. 


PROTEINS.  83 

gradually  passes  into  a  deep  wine  red.     Usually  black,  amorphous  par- 
ticles separate  out  and  the  solution  becomes  turbid. 

The  reaction  cannot  be  obtained  in  solutions  containing  free  alkali.  It 
is  best  to  use  such  an  amount  of  bromine  as  will  produce  a  permanent 
yellow  color  in  the  cold.  The  use  of  a  less  amount  of  bromine  than  this 
produces  a  weak  coloration  whereas  an  excess  of  bromine  prevents  the 
reaction.  The  test  is  not  v^ry  delicate,  but  a  characteristic  reaction  may 
always  be  obtained  in  1:1000  solutions.  The  only  histidine  derivative 
which  yields  a  similar  coloration  is  imidazolethylamine,  and  the  reaction 


\C£ 


Fig.  26. — Histidine  Bichloride. 

in  this  case  is  rather  weak  as  compared  with  the  color  obtained  with 
histidine  or  histidine  salts. 

Valine,  C-Hj^NOj. — The  amino- valerianic  acid  obtained  from 
proteins  is  a-amino-isovalerianic  acid,  and  as  such  bears  the  following 
formula : 

CH3    NH2 

H-C C-COOH. 

1  I 

CH3   H 

It  closely  resembles  leucine  in  many  of  its  properties,  but  is  more  soluble 
in  water.     It  is  a  difficult  matter  to  identify  valine  in  the  presence  of 
leucine  and  isoleucine  inasmuch  as  these  amino  acids  crystallize  together 
in  such  a  way  that  the  combination  persists  even  after  repeated  recrystal- . 
lizations.     Valine  is  dextro-rotatory. 

Arginine,  CgHj^N^Oj. — Arginine  is  guanidine-a-amino-valerianic 
acid  and  possesses  the  following  structural  formula: 


84  PHYSIOLOGICAL   CHEMISTRY. 

H    H    H    NH^ 

NH-C-C-C-C-COOH. 

I  1      I       I       I 

NH=C         H    H    H    H 

It  has  been  obtained  from  every  protein  so  far  subjected  to  decomposition. 
The  arginine  obtained  from  proteins  is  dextro-iotatory,  and  has  pro- 
nounced basic  properties,  reacts  strongly  alkaline  to  litmus,  and  forms 
stable  carbonates.  Because  of  these  facts,  Kossel  considers  arginine  to  be 
the  nucleus  of  the  protein  molecule.  It  is  obtained  in  widely  different 
amounts  from  different  proteins,  over  85  per  cent  of  certain  protamines 
having  been  obtained  in  the  form  of  this  amino  acid.  It  is  claimed  that  in 
the  ordinary  metabolic  activities  of  the  animal  body  arginine  gives  rise 
to  urea.  While  this  claim  is  probably  true,  it  should,  at  the  same  time, 
be  borne  in  mind  that  the  greater  part  of  the  protein  nitrogen  is  eliminated 
as  urea  and  that,  therefore,  but  a  very  small  part  can  arise  from  arginine. 
Leucine,  CgH^gNOg. — Leucine  is  an  abundant  end-product  of  the 
decomposition  of  protein  material,  and  was  one  of  the  first  of  these 
products  to  be  discovered.  It  is  a-amino-isohutyl-acetic  acid,  and 
therefore  has  the  following  formula: 

CH3      NH2 
H-C-CH^-C-COOH. 
CH3      H 

The  leucine  which  results  from  protein  decomposition  is  /-leucine. 
Leucine  is  present  normally  in  the  pancreas,  thymus,  thyroid,  spleen,  brain, 
liver,  kidneys,  and  salivary  glands.  It  has  been  found  pathologically  in 
the  urine  (in  acute  yellow  atrophy  of  the  liver,  in  acute  phosphorus 
poisoning,  and  in  severe  cases  of  typhoid  fever  and  smallpox),  and  in 
the  liver,  blood,  and  pus. 

Pure  leucine  crystallizes  in  thin,  white,  hexagonal  plates.  Crystals  of 
pure  leucine  are  reproduced  in  Fig.  27.  It  is  rather  easily  soluble  in  water 
(46  parts),  alkalis,  ammonia,  and  acids.  On  rapid  heating  to  295°  C, 
leucine  decomposes  with  the  formation  of  carbon  dioxide,  ammonia,  and 
amylamine.  Aqueous  solutions  of  leucine  obtained  from  proteins  are  laevo- 
rotatory,  but  its  acid  or  alkaline  solutions  are  dextro-rotatory.  So-called 
impure  leucine^  is  a  slightly  refractive  substance,  which  generally  crystal- 

*  These  balls  of  so-called  impure  leucine  do  contain  considerable  leucine,  but  inasmuch 
as  they  may  contain  many  other  things  it  is  a  bad  practice  to  allude  to  them  as  leucine. 


PROTEINS. 


85 


lizes  in  balls  having  a  radial  structure,  or  in  aggregations  of  spherical 
bodies,  Fig.  109,  Chapter  XX. 

Isoleucine,  CgHjgNOj. — Isoleucine  is  a-amino-^-methyl-^-elhyl-pro- 
pionic  acid,  and  possesses  the  following  structural  formula: 


CIL 


H-C 


C2H5 


NH, 

C-COOH. 

I 
H 


This  amino  acid  was  discovered  by  Ehrlich  in  1903.     Its  presence  has 
been  established  among  the  decomposition  products  of  only  a  few  proteins 


Fig.  27. — Leucine. 

although  it  probably  occurs  among  those  of  many  or  most  of  them.  Ehr- 
lich has  shown  that  the  d-o^myl  alcohol  which  is  produced  by  yeast  fermen- 
tation originates  from  isoleucine  and  the  isoamylalcohol  originates  from 
leucine.     Isoleucine  is  dextro-rotatory. 

Lysine,  CgHi^NjOg. — The  three  bodies,  lysine,  arginine,  and  his- 
tidine,  are  frequently  classed  together  as  the  hexone  bases.  Lysine  was 
the  first  of  the  bases  discovered.  It  is  a-e-diainino-caproic  acid  and  hence 
possesses  the  following  structure: 

NH^H  H    H    NH, 

I      !     I     I     I 

H-C-C-C-C-C-COOH 

i        I      I       I      I 
H     H    H    H    H 

It  is  dextro-rotatory  and  is  found  in  relatively  large  amount  in  casein  and 
gelatin.  Lysine  is  obtained  from  nearly  all  proteins,  but  is  absent  from 
the  vegetable  proteins  which  are  soluble  in  strong  alcohol.  It  is  the 
mother-substance  of  cadaverin  and  has  never  been  obtained  in  crystalline 


PHYSIOLOGICAL   CHEMISTRY. 


form.     Lysine  is  usually  obtained  as  the  picrate  which  is  sparingly  soluble 
in  water  and  crystallizes  readily.     These  crystals  are  shown  in  Fig.  28. 


Fig.  28. — Lysine  Picrate. 


Fig.  29. — AsPARTic  Acid. 

Aspartic  Acid,  C^H^NO^. — Aspartic  acid  is  amino-succinic  acid  and 
has  the  following  structural  formula: 

H-CCOOH 
H-CCOOH. 


H 

The  amide  of  aspartic  acid,  asparagine,  is  very  widely  distributed  in 
the  vegetable  kingdom.  The  crystalline  form  of  aspartic  acid  is  exhibited 
in  Fig.  29. 


PROTEINS. 


87 


Aspartic  acid  has  been  found  among  the  decomposition  products  of 
all  the  proteins  examined,  except  the  protamines.  It  has  not  been  obtained, 
however,  in  very  large  proportion  from  any  of  them.  The  aspartic  acid 
obtained  from  protein  is  laevo-rotatory. 

Glutamic  Acid,  C^HgNO^. — This  acid  is  a-amino-normal-glutaric 
acid  and  as  such  bears  the  following  graphic  formula : 

NH, 

I 
H-CCOOH 

I 
H-C-H 

I 
H-CCOOH. 

H 

Glutamic  acid  is  yielded  by  all  the  proteins  thus  far  examined,  except 
the  protamines,  and  by  most  of  these  in  larger  amount  than  any  other  of 


Fig.  30. — Glutamic  Acid. 
Reproduced  from  a  micro-photograph  made  by  Prof.  E.  T.  Reichert,  of   the  University  of 

Pennsylvania. 

their  decomposition  products.  It  is  yielded  in  especially  large  proportion 
by  most  of  the  proteins  of  seeds,  43.66  per  cent  ha^•ing  been  obtained 
very  recently  by  Osborne  and  Guest  ^  by  the  hydrolysis  of  gliadin,  the 
prolamin  of  wheat.  This  is  the  largest  amount  of  any  single  decompo- 
sition product  yet  obtained  from  any  protein  except  the  protamines. 

Glutamic  acid  and  aspartic  acid  are  the  only  dibasic  acids  which  have 
thus  far  been  obtained  as  decomposition  products  of  proteins.  As  there 
is  an  apparent  relation  between  the  proportion  of  these  acids  and  that  of 

'  Osborne  and  Guest;  Jour.  Biol.  Ghent.,  9,  425,  igii 


88 


PHYSIOLOGICAL    CHEMISTRY. 


ammonia  which  the  different  proteins  yield  it  is  possible  that  one  of  the 
carbox}'!  groups  of  these  acids  is  united  with  NH2  as  an  amide,  the  other 
carboxyl  group  being  united  in  polypeptide  union  (see  page  75)  with  some 
other  amino  acid.     This  might  be  represented  by  the  following  formula: 

R-CHNH-COOH 

/ 
CO-  CHXH3-  CH.-  CH,-  CONH2. 

It  has  not  been  definitely  proven,  however,  that  this  form  of  linking 
actually  occurs. 

The  glutamic  acid,  }delded  by  proteins  upon  hydrolysis,  is  dextro- 
rotatory.    Crystals  of  glutamic  acid  are  reproduced  in  Fig.  30,  p.  87. 


Fig.  31.— L^vo-a-PROLENT;. 


Proline,    CgHgNOg. — ^Proline   is    a-pyrroUdine-carhoxylic  acid    and 
possesses  the  following  graphic  structure: 


-CH, 


H,C- 

H^C^^/CHCOOH. 
NH 

Proline  was  first  obtained  as  a  decomposition  product  of  casein.  Proline 
obtained  from  proteins  is  laevo-rotatory  and  is  the  only  protein  decomposi- 
tion product  which  is  readily  soluble  in  alcohol.  It  is  also  one  of  the  few 
heterocyclic  compounds  obtained  from  proteins.  Prohne  has  been  found 
among  the  decomposition  products  of  all  proteins  except  the  protamines. 
The  maximum  yield  reported  is  13.73  per  cent  obtained  by  Osborne  and 
Clapp   from   the   hydrolysis   of   hordein.     More   recently  Fischer   and 


PROTEINS. 


89 


Boehner*  reported  having  obtained  7.7  per  cent  from  the  hydrolysis  of 
gelatin.  The  crystalline  form  of  Icrvo- a- proline  is  shown  in  Fig.  31,  and 
the  copper  salt  of  proline  is  represented  by  a  micro-photograph  in  Fig.  32, 
below.  The  crystals  of  the  copper  salt  have  a  deep  blue  color,  but  when 
they  lose  their  water  of  crystallization  they  assume  a  characteristic  violet 
color. 


-\^^ 


Fig.  32. — Copper  Salt  of  Proline. 
Reproduced  from  a  micro- photograph  made  by  Prof.  E.  T.  Reichert,  of  the  Universit}-  of 

Pennsylvania. 

Oxyproline,  C5H9XO3. — Oxyproline  was  discovered  by  Fischer. 
It  has  as  yet  been  obtained  from  only  a  few  proteins,  but  this  may  be  due 
to  the  fact  that  only  a  few  have  been  examined  for  its  presence.  The 
position  of  the  hydroxyl  group  has  not  yet  been  established. 

Diaminotrihydroxydodecanoic  Acid,  Ci2H2gN,05. — This  amino 
acid  was  discovered  by  Fischer  and  Abderhalden  as  a  product  of  the 
hydrolysis  of  casein.  It  has  thus  far  been  obtained  from  no  other  source. 
It  is  laevo-rotatorv  and  its  constitution  has  not  been  determined. 


Experiments. 

While  the  ordinary  courses  in  physiological  chemistry  preclude  any 
extended  study  of  the  decomposition  products  of  proteins,  the  manipula- 
tion of  a  simple  decomposition  and  the  subsequent  isolation  and  study  of  a 
few  of  the  products  most  easily  and  quickly  obtained  will  not  be  without 
interest.^     To  this  end  the  student  may  use  the  following  decomposition 

*  Fischer  and  Boehner:  Zeit.  phys.  client.,  65,  p.  118,  1910. 

*  The  procedure  here  set  forth  has  nothing  in  common  with  the  procedure  by  means 
of  which  the  long  line  of  decomposition  products  just  enumerated  are  obtained.  This  latter 
process  is  an  exceedingly  compUcated  one  which  is  entirely  outside  the  province  of  any  course 
in  physiological  chemistry. 


90  PHYSIOLOGICAL   CHEMISTRY. 

procedure :  Treat  the  protein  in  a  large  flask  with  water  containing  3-5 
per  cent  of  H^SO^  and  place  it  on  a  water-bath  until  the  protein  material 
has  been  decomposed  and  there  remains  a  fine,  fluffy,  insoluble  residue. 
Filter  off  this  residue  and  neutralize  the  filtrate  with  Ba(0H)2  and  BaCOg. 
Filter  off  the  precipitate  of  BaSO^  which  forms  and  when  certain  that 
the  fluid  is  neutral  or  faintly  acid,  ^  concentrate  (first  on  a  wire  gauze  and 
later  on  a  water-bath)  to  a  syrup.  This  syrup  contains  the  end-products 
of  the  decomposition  of  the  protein,  among  which  are  proteoses,  peptones, 
tyrosine,  leucine,  etc.  Add  95  per  cent  alcohol  slowly  to  the  warm  syrup 
until  no  more  precipitate  forms,  stirring  continuously  with  a  glass  rod. 
This  precipitate  consists  of  proteoses  and  peptones.  Gather  the  sticky 
precipitate  on  the  rod  or  the  sides  of  the  dish,  and,  after  warming  the 
solution  gently  for  a  few  moments,  filter  it  through  a  filter  paper  which  has 
not  been  previously  moistened.  After  dissolving  the  precipitate  of  pro- 
teoses and  peptones  in  water^  the  solution  may  be  treated  according  to 
the  method  of  separation  given  on  page  120. 

The  leucine  and  tyrosine,  etc.,  are  in  solution  in  the  warm  alcoholic 
filtrate.  Concentrate  this  filtrate  on  the  water-bath  to  a  thin  syrup, 
transfer  it  to  a  beaker,  and  allow  it  to  stand  over  night  in  a  cool  place  for 
crystallization.  The  tyrosine  first  crystallizes  (Fig.  24,  page  81),  followed 
later  by  the  formation  of  characteristic  crystals  of  impure  leucine  (see  Fig. 
109,  Chapter  XX).  After  examining  these  crystals  under  the  microscope, 
strain  off  the  crystalline  material  through  fine  muslin,  heat  it  gently  in  a 
little  water  to  dissolve  the  leucine  (the  tyrosine  will  be  practically  insoluble) 
and  filter.  Concentrate  the  filtrate  and  allow  it  to  stand  in  a  cool  place 
over  night  for  the  crude  leucine  to  crystallize.  Filter  off  the  crystals  and 
use  them  in  the  tests  for  leucine  given  on  page-9i.  The  crystals  of  tyrosine 
remaining  on  the  paper  from  the  first  filtration  may  be  used  in  the  tests  for 
tyrosine  as  given  below.  If  desired,  the  tyrosine  and  leucine  may  be 
purified  by  recry stallizing  in  the  usual  manner.  Habermann  has  suggested 
a  method  of  separating  leucine  and  tyrosine  by  means  of  glacial  acetic  acid. 

Experiments  on  Tyrosine.    . 

Make  the  following  tests  with  the  tyrosine  crystals  already  prepared 
or  upon  some  pure  tyrosine  furnished  by  the  instructor. 

I.  Microscopical  Examination. — ^Place  a  minute  crystal  of  tyrosine 
on  a  slide,  add  a  drop  of  water,  cover  with  a  coverglass,  and  examine 

'  If  the  solution  is  alkaline  in  reaction  at  this  point,  the  amino  acids  will  be  broken  down 
and  ammonia  will  be  evolved. 

^  At  this  point  the  aqueous  solution  of  the  proteoses  and  peptones  may  be  filtered  to  remove 
any  BaSO^  which  may  still  remain.  Tyrosine  crystals  will  also  be  found  here,  since  it  is 
less  soluble  than  the  leucine  and  may  adhere  to  the  proteose-peptone  precipitate.  Add  the 
cr}'stals  of  tyrosine  to  the  warm  alcohol  filtrate. 


PROTEINS.  91 

microscopically.  Now  run  more  water  under  the  cover-glass  and  warm 
in  a  bunsen  flame  until  the  tyrosine  has  dissolved.  Allow  the  solution  to 
cool  slowly,  then  examine  again  microscopically,  and  compare  the  crystals 
with  those  shown  in  Fig.  24,  page  81. 

2.  Solubility. — Try  the  solubility  of  very  small  amounts  of  tyrosine  in 
cold  and  hot  water,  cold  and  hot  95  per  cent  alcohol,  dilute  NH^OH, 
dilute  KOH  and  dilute  HCl. 

3.  Sublimation. — Place  a  little  tyrosine  in  a  dry  test-tube,  heat 
gently  and  notice  that  the  material  does  not  sublime.  How  does  this 
compare  with  the  result  of  Experiment  3  under  Leucine  ? 

4.  Hoffman's  Reaction. — This  is  the  name  given  to  Millon's  reaction 
when  employed  to  detect  tyrosine.  Add  about  3  c.c.  of  water  and  a  few 
drops  of  Millon's  reagent  to  a  little  tyrosine  in  a  test-tube.  Upon  dissolv- 
ing the  tyrosine  by  heat  the  solution  gradually  darkens  and  may  assume  a 
dark  red  color.     What  group  does  this  test  show  to  be  present  in  tyrosine  ? 

5.  Piria's  Test. — Warm  a  little  tyrosine  on  a  watch  glass  on  a  boiling 
water-bath  for  20  minutes  w'ith  3-5  drops  of  cone.  HjSO^.  Tyrosine- 
sulphuric  acid  is  formed  in  the  process.  Cool  the  solution  and  wash  it 
into  a  small  beaker  with  water.  Now  add  CaCOg  in  substance  slowly 
v\ath  stirring,  until  the  reaction  of  the  solution  is  no  longer  acid.  Filter, 
concentrate  the  filtrate,  and  add  to  it  a  few  drops  (avoid  an  excess)  of 
very  dilute  neutral  ferric  chloride.  A  purple  or  violet  color,  due  to  the 
formation  of  the  ferric  salt  of  tyrosine-sulphuric  acid,  is  produced.  This 
is  one  of  the  most  satisfactory  tests  for  the  identification  of  tyrosine. 

6.  Morner's  Test. — Add  about  3  c.c.  of  Morner's  reagent^  to  a  little 
tyrosine  in  a  test-tube,  and  gently  raise  the  temperature  to  the  boiling- 
point.     A  green  color  results. 

Experiments  ox  Leucine. 

Make  the  following  test  upon  the  leucine  crystals  already  prepared  or 
upon  some  pure  leucine  furnished  by  the  instructor. 

I,  2  and  3.  Repeat  these  experiments  according  to  the  directions 
given  under  Tyrosine  (pages  90  and  91). 

'  Morner's  reagent  is  prepared  by  thoroughly  mixing  i  volume  of  formalin,  45  volumes 
of  distilled  water,  and  55  volumes  of  concentrated  sulphuric  acid. 


CHAPTER  V. 

PROTEINS  :  THEIR  CLASSIFICATION  AND 
PROPERTIES. 

From  what  has  already  been  said  in  Chapter  IV  regarding  the  protein 
substances  it  will  be  recognized  that  the  grouping  of  the  diverse  forms  of 
this  class  of  substances  in  a  logical  manner  is  not  an  easy  task.  The  fats 
and  carbohydrates  may  be  classified  upon  the  fundamental  principles  of 
their  stereo-chemical  relationships,  whereas  such  a  system  of  classification 
in  the  case  of  the  proteins  is  absolutely  impossible  since,  as  we  have 
already  stated,  the  molecular  structure  of  these  complex  substances  is 
unknown.  Because  of  the  diversity  of  standpoint  from  which  the  proteins 
may  be  viewed,  relative  to  their  grouping  in  the  form  of  a  logically  classi- 
fied series,  it  is  obvious  that  there  is  an  opportunity  for  the  presentation 
of  classifications  of  a  widely  divergent  character.  The  fact  that  there 
were  until  recently  at  least  a  dozen  different  classifications  which  were 
recognized  by  various  groups  of  English-speaking  investigators  emphasizes 
the  difficulties  in  the  way  of  the  individual  or  individuals  who  would 
offer  a  classification  which  should  merit  universal  adoption.  Realizing 
the  great  handicap  and  disadvantage  which  the  great  diversity  of  the 
protein  classifications  was  forcing  upon  the  workers  in  this  field,  the 
Chemical  and  Physiological  Societies  of  England  recently  drafted  a  classi- 
fication which  appealed  to  these  groups  of  scientists  as  fulfilling  all  require- 
ments and  presented  it  for  the  consideration  of  the  American  Physiological 
Society  and  the  American  Society  of  Biological  Chemists.  The  outcome 
of  this  has  been  that  there  are  now  only  two  protein  classifications  which 
are  recognized  by  English-speaking  scientists,  one  the  British  Classification, 
the  other  the  American  Classification.  These  classifications  are  very 
similar  and  doubtless  will  ultimately  be  merged  into  a  single  classification. 
In  our  consideration  of  the  proteins  we  shall  conform  in  all  details  to  the 
American  Classification.  In  this  connection  we  will  say,  however,  that 
we  feel  that  the  English  Societies  have  strong  grounds  for  preferring  the 
use  of  the  term  scleroproteins  for  albuminoids  and  chromoproteins  for 
heemoglobins.     The  two  classifications  are  as  follows: 

92 


PROTEINS.  93 

CLASSIFICATION  OF  PROTEINS  ADOPTED  BY  THE  AMERI- 
CAN   PHYSIOLOGICAL    SOCIETY    AND    THE    AMERICAN 
SOCIETY  OF  BIOLOGICAL  CHEMISTS. 

I.  SIMPLE  PROTEINS. 

Protein  substances  which  yield  only  a-amino  acids  or  their  derivatives 
on  hydrolysis. 

(a)  Albumins. — Soluble  in  pure  water  and  coagulable  by  heat, 
e.  g.,  ovalbumin,  serum  albumin,  lactalbumin,  vegetable  albumins. 

(b)  Globulins. — Insoluble  in  pure  water  but  soluble  in  neutral 
solutions  of  salts  of  strong  bases  with  strong  acids/  e.  g.,  serum  globulin, 
ovo globulin,  edeslin,  amandin,  and  other  vegetable  globulins. 

(c)  Glutelins. — Simple  proteins  insoluble  in  all  neutral  solvents,  but 
readily  soluble  in  very  dilute  acids  and  alkalis/  e.  g.,  glutenin. 

id)  Alcohol-soluble  Proteins  (Prolamins).^ — Simple  proteins  sol- 
uble in  70-So  per  cent  alcohol,  insoluble  in  water,  absolute  alcohol,  and 
other  neutral  solvents,*  e.g.,  zein,  gliadin,  hordein,  and  bynin. 

ie)  Albuminoids. — Simple  proteins  possessing  a  similar  structure  to 
those  already  mentioned,  but  characterized  by  a  pronounced  insolubility 
in  all  neutral  solvents,^  e.g.,  elastin,  collagen,  keratin. 

(J)  Histones. — Soluble  in  water  and  insoluble  in  very  dilute  ammo- 
nia, and,  in  the  absence  of  ammonium  salts,  insoluble  even  in  excess  of 
ammonia;  yield  precipitates  with  solutions  of  other  proteins  and  a  coagu- 
lum  on  heating  which  is  easily  soluble  in  very  dilute  acids.  On  hydrolysis 
they  yield  a  large  number  of  amino  acids  among  which  the  basic  ones  pre- 
dominate. In  short,  histones  are  basic  proteins  which  stand  between 
protamines  and  true  proteins,  e.g.,  globin,   thymus  histone,  scombrone. 

(g)  Protamines. — Simpler  polypeptides  than  the  proteins  included 
in  the  preceding  groups.  They  are  soluble  in  water,  uncoagulable  by 
heat,  have  the  property  of  precipitating  aqueous  solutions  of  other  pro- 
teins, possess  strong  basic  properties  and  form  stable  salts  with  strong 
mineral  acids.  They  yield  comparatively  few  amino  acids,  among  which 
the  basic  ones  predominate.  They  are  the  simplest  natural  proteins,  e.  g., 
salmine,  sturine,  clupeine,  scombrine. 

'  The  precipitation  limits  with  ammonium  sulphate  should  not  be  made  a  basis  for  dis- 
tinguishing the  albumins  from  the  globulins. 

-'  Such  substances  occur  in  abundance  in  the  seeds  of  cereals  and  doubtless  represent  a 
well-defined  natural  group  of  simple  proteins. 

'  The  name  prolamins  has  been  suggested  for  these  alcohol-soluble  proteins  by  Dr.  Thomas 
B.  Osborne  (Science,  iqoS,  XX\'III,  p.  417).  It  is  a  very  fitting  term  inasmuch  as  upon 
hydrolysis  they  yield  particularly  large  amounts  of  proline  and  ammonia. 

*  The  subclasses  defined  (a,  b,  c,  d,)  are  exemplified  by  proteins  obtained  from  both  plants 
and  animals.  The  use  of  appropriate  prefixes  will  suffice  to  indicate  the  origin  of  the  com- 
pounds, e.  g.,  tnoglobuiin,  laclalhumin,  etc. 

^  These  form  the  principal  organic  constituents  of  the  skeletal  structure  of  animals  and  also 
their  external  covering  and  its  appendages.  This  definition  does  not  provide  for  gelatin 
which  is,  however,  an  artificial  derivative  of  collagen. 


94  PHYSIOLOGICAL    CHEMISTRY. 

II.  CONJUGATED  PROTEINS. 

Substances  which  contain  the  protein  molecule  united  to  some  other 
molecule  or  molecules  otherwise  than  as  a  salt. 

{a)  Nucleoproteins. — Compounds  of  one  or  more  protein  molecules 
with  nucleic  acid,  e.  g.,  cy  to  globulin,  nucleohistone. 

(b)  Glycoproteins. — Compounds  of  the  protein  molecule  with  a 
substance  or  substances  containing  a  carbohydrate  group  other  than  a 
nucleic  acid.  e.  g.,  mucins  and  mucoids  {osseomucoid,  iendomucoid,  sero- 
mucoid,  ichthulin,  helicoprotein). 

(c)  Phosphoproteins. — Compounds  of  the  protein  molecule  with 
some,  as  yet  undefined,  phosphorus-containing  substances  other  than  a 
nucleic  acid  or  lecithin,^  e.  g.,  caseinogen,  vitellin. 

(d)  Haemoglobins. — Compounds  of  the  protein  molecule  with 
haematin,  or  some  similar  substance,  e.g.,  hcemoglobin,  hcEmocyanin. 

{e)  Lecithoproteins. — Compounds  of  the  protein  molecule  with 
lecithins,  e.  g.,  lecithans,  phosphatides. 

III.  DERIVED  PROTEINS. 

I.  Primary  Protein  Derivatives. 

Derivatives  of  the  protein  molecule  apparently  formed  through 
hydrolytic  changes  which  involve  only  slight  alteration  of  the  protein 
molecule. 

{a)  Proteans. — Insoluble  products  which  apparently  result  from 
the  incipient  action  of  water,  very  dilute  acids  or  enzymes,  e.  g.,  myosan, 
edesian. 

(b)  Metaproteins. — ^Products  of  the  further  action  of  acids  and  alkalis 
whereby  the  molecule  is  so  far  altered  as  to  form  products  soluble  in  very 
weak  acids  and  alkalis  but  insoluble  in  neutral  fluids,  e.g.,  acid  meta- 
protein  {acid  albuminate) ,  alkali  metaprotein  {alkali  albuminate). 

ic)  Coagulated  Proteins.— Insoluble  products  which  result  from 
(i)  the  action  of  heat  on  their  solutions,  or  (2)  the  action  of  alcohol  on  the 
protein. 

2.  Secondary  Protein  Derivatives.^ 

Products  of  the  further  hydrolytic  cleavage  of  the  protein  molecule. 

{a)  Proteoses. — Soluble  in  water,  non-coagulable  by  heat,  and 
precipitated  by  saturating  their  solutions  with  ammonium — or  zinc 
sulphate,'  e.  g.,  protoproteose,  deuteroproteose. 

*  The  accumulated  chemical  evidence  distinctly  points  to  the  propriety  of  classifying 
the  phosphoproteins  as  conjugated  compounds,  i.  e.,  they  are  possibly  esters  of  some  phosphoric 
acid  or  acids  and  protein. 

^  The  term  secondary  protein  derivatives  is  used  because  the  formation  of  the  primary 
derivatives  usually  precedes  the  formation  of  the  secondary  derivatives. 

'  As  thus  defined,  this  term  does  not  strictly  cover  all  the  protein  derivatives  commonly 
called  proteoses,  e.  g.,  heteroproteose  and  dysproteose. 


PROTEINS.  95 

{b)  Peptones. — Soluble  in  water,  non-coagulable  by  heat,  but  not 
precipitated  by  saturating  their  solutions  with  ammonium  sulphate/  e.  g., 
antipeptaue,  a  mpho peptone. 

(c)  Peptides. — Defmitely  characterized  combinations  of  two  or  more 
amino  acids,  the  carboxyl  group  of  one  being  united  with  the  amino  group 
of  the  other  with  the  elimination  of  a  molecule  of  water, ^  e.  g.,  dipeptides, 
tripeptides,  tetrapeptides,  pentapeptides. 

CLASSIFICATION   OF   PROTEINS   ADOPTED   BY  THE    CHEM- 
ICAL AND  PHYSIOLOGICAL  SOCIETIES 
OF  ENGLAND. 

I.  Simple  Proteins. 

1.  Protamines,  e.g.,  salmine,  clupeine. 

2.  Histones,  e.  g.,  globin,  scombrone. 

3.  Albumins,  e.g.,  ovalbumin,  serum  albumin,  vegetable  albumins. 

4.  Globulins,  e.g.,  serum  globulin,  ovoglobulin,  vegetable  globulins. 

5.  Glutelins,  e.  g.,  glutenin. 

6.  Alcohol-soluble  proteins,  e.  g.,  zein,  gliadin. 

7.  Scleroproteins,  e.  g.,  elastin,  keratin. 

8.  Phosphoproteins,  e.g.,  caseinogen,  vitellin. 

II.  Conjugated  Proteins. 

1.  Glucoproteins,  e.g.,  mucins,  mucoids. 

2.  Nucleoproteins,  e.  g.,  nucleohistone,  cyto globulin. 

3.  Chromoproteins,  e.  g.,  hamo globin,  hcemocyanin. 

III.  Products  of  Protein  Hydrolysis. 

1.  Infraproteins,  e.  g.,  acid  infraprotein  {acid  albuminate),  alkali 
infraprotein  {alkali  albuminate) . 

2.  Proteoses,  e.g.,  protoproteose,  heteroproteose,  deuteroproteose. 

3.  Peptones,  e.g.,  amphopeptone,  antipeptone. 

4.  Polypeptides,  e.g..  dipeptides,  tripeptides,  tetrapeptides. 

CONSIDERATIONS  OF  THE  VARIOUS  CLASSES 
OF  PROTEINS. 

SIMPLE  PROTEINS. 

The  simple  proteins  are  true  protein  substances  which,  upon  hy- 
drolysis,   yield   only    a-amino    acids    or   their   derivatives.     "Although 

'  In  this  group  the  kjTines  may  be  included.  For  the  present  it  is  believed  that  it  will 
be  helpful  to  retain  this  term  as  defined,  reserving  the  expression  peptide  for  the  simpler 
compounds  of  definite  structure,  such  as  dipeptides,  etc. 

^  The  peptones  are  undoubtedly  peptides  or  mixtures  of  peptides,  the  latter  term  being 
at  present  used  to  designate  those  of  definite  structure. 


96  PHYSIOLOGICAL    CHEMISTRY. 

no  means  are  at  present  available  whereby  the  chemical  individuality  of 
any  protein  can  be  established,  a  number  of  simple  proteins  have  been 
isolated  from  animal  and  vegetable  tissues  which  have  been  so  well 
characterized  by  constancy  of  ultimate  composition  and  uniformity  of 
physical  properties  that  they  may  be  treated  as  chemical  individuals 
until  further  knowledge  makes  it  possible  to  characterize  them  more 
definitely."  Under  simple  proteins  we  may  class  albumins,  globulins, 
glutelins,  prolamins,  albuminoids,  histones  and  protamines. 

ALBUMINS. 
Albumins  constitute  the  first  class  of  simple  proteins  and  may  be 
defined  as  simple  proteins  which  are  coagulable  by  heat  and  soluble 
in  pure  (salt-free)  water.  Those  of  animal  origin  are  not  precipitated 
upon  saturating  their  neutral  solutions  at  30°  C.  with  sodium  chloride 
or  magnesium  sulphate,  but  if  a  saturated  solution  of  this  character 
be  acidified  with  acetic  acid  the  albumin  precipitates.  All  albumins 
of  animal  origin  may  be  precipitated  by  saturating  their  solutions  with 
ammonium  sulphate.^  They  may  be  thrown  out  of  solution  by  the 
addition  of  a  sufficient  quantity  of  a  mineral  acid,  whereas  a  weak 
acidity  produces  a  slight  precipitate  which  dissolves  'upon  agitating  the 
solution.  Metallic  salts  also  possess  the  property  of  precipitating  al- 
bumins, some  of  the  precipitates  being  soluble  in  excess  of  the  reagent, 
whereas  others  are  insoluble  in  such  an  excess.  Of  those  proteins 
which  occur  native  the  albumins  contain  the  highest  percentage  of  sul- 
phur, ranging  from  1.6  to  2.5  per  cent.  Some  albumins  have  been 
obtained  in  crystalline  form,  notably  egg  albumin,  serum  albumin,  and 
lactalbumin,  but  the  fact  that  they  may  be  obtained  in  crystalline  form 
does  not  necessarily  prove  them  to  be  chemical  individuals. 

GENERAL  COLOR  REACTIONS  OF  PROTEINS. 

These  color  reactions  are  due  to  a  reaction  between  some  one  or 
more  of  the  constituent  radicals  or  groups  of  the  complex  protein  molecule 
and  the  chemical  reagent  or  reagents  used  in  any  given  test.  Not  all 
proteins  contain  the  same  groups  and  for  this  reason  the  various  color 
tests  will  yield  reactions  varying  in  intensity  of  color  according  to  the 
nature  of  the  groups  contained  in  the  particular  protein  under  examina- 
tion. Various  substances  not  proteins  respond  to  certain  of  these  color 
reactions,  and  it  is  therefore  essential  to  submit  the  material  under  ex- 
amination to  several  tests  before  concluding  defmitely  regarding  its 
nature. 

'  In  this  connection,  Osborne's  observation  that  there  are  certain  vegetable  albumins 
which  are  precipitated  by  saturating  their  solutions  with  sodium  chloride  or  magnesium 
sulphate  or  by  half-saturating  with  ammonium  sulphate,  is  of  interest. 


PROTEINS.  97 

TECHNIC  OF  THE  COLOR  REACTIONS. 

1.  Millon's  Reaction. — To  5  c.c.  of  a  dilute  solution  of  egg  aK 
bumin  in  a  test-tube  add  a  few  drops  of  Millon's  reagent.  A  white 
precipitate  forms  which  turns  red  when  heated.  This  test  is  a  partic- 
ularly satisfactory  one  for  use  on  solid  proteins,  in  which  case  the  reagent 
is  added  directly  to  the  solid  substance  and  heat  applied,  which  causes 
the  substance  to  assume  a  red  color.  Such  proteins  as  are  not  precipitated 
by  mineral  acids,  for  example  certain  of  the  proteoses  and  peptones, 
yield  a  red  solution  instead  of  a  red  precipitate. 

The  reaction  is  due  to  the  presence  of  the  hydroxy-phenyl  group, 
— CgH^OH,  in  the  protein  molecule  and  certain  non-proteins  such  as 
tyrosine,  phenol  (carbolic  acid)  and  thymol  also  respond  to  the  reaction. 
Inasmuch  as  the  tyrosine  grouping  is  the  only  hydroxyphenyl  grouping 
which  has  definitely  been  proven  to  be  present  in  the  protein  molecule  it 
is  e^ident  that  protein  substances  respond  to  Millon's  reaction  because 
of  the  presence  of  this  tyrosine  complex.  The  test  is  not  a  very  satis- 
factory one  for  use  in  solutions  containing  inorganic  salts  in  large  amount, 
since  the  mercury  of  the  Millon's  reagent^  is  thus  precipitated  and  the 
reagent  rendered  inert.  This  reagent  is  therefore  never  used  for  the 
detection  of  protein  material  in  the  urine. 

2.  Xanthoproteic  Reaction. — To  2-3  c.c.  of  egg  albumin  solu- 
tion in  a  test-tube  add  concentrated  nitric  acid.  A  white  precipitate 
forms,  which  upon  heating  turns  yellow  and  finally  dissolves,  imparting 
to  the  solution  a  yellow  color.  Cool  the  solution  and  carefully  add 
ammonium  hydroxide,  potassium  hydroxide,  or  sodium  hydroxide  in 
excess.  Note  that  the  yellow  color  deepens  into  an  orange.  This 
reaction  is  due  to  the  presence  in  the  protein  molecule  of  the  phenyl 
group,  with  which  the  nitric  acid  forms  certain  nitro  modifications. 
The  particular  complexes  of  the  protein  molecule  which  are  of  especial 
importance  in  this  connection  are  those  of  tyrosine,  phenylalanine,  and 
tryptophane.  The  test  is  not  a  satisfactory  one  for  use  in  urinary  ex- 
amination because  of  the  color  of  the  end-reaction. 

3.  Adamkiewicz  Reaction. — Thoroughly  mix  i  volume  of  con- 
centrated sulphuric  acid  and  2  volumes  of  acetic  acid  in  a  test-tube, 
add  a  few  drops  of  egg  albumin  solution  and  heat  gently.  A  reddish- 
violet  color  is  produced.  Gelatin  does  not  respond  to  this  test.  This 
reaction  shows  the  presence  of  the  tryptophane  group  (see  next  experi- 
ment). The  test  depends  upon  the  presence  of  glyoxylic  acid,  CHO.- 
C00H4-H,0  or   CH(OH)XOOH,  in  the  reagents.     This  is    shown 

'  Millon's  reagent  consists  of  mercury  dissolved  in  nitric  acid  containing  some  nitrous 
acid.  It  is  prepared  by  digesting  one  part  (by  weight)  of  mercury  with  two  parts  (by  weight) 
of  HNOj  (sp.  gr.  1.42)  and  diluting  the  resulting  solution  with  two  volumes  of  water. 


98  PHYSIOLOGICAL    CHEMISTRY. 

by  the  failure  to  secure  a  positive  reaction  when  acetic  acid  free  from 
glyoxyhc  acid  is  used. 

Rosenheim  has  recently  advanced  the  view  that  the  reaction  may 
be  due  to  the  presence  of  oxidizing  agents  such  as  nitrous  acid  and 
ferric  salts  in  the  sulphuric  acid. 

4.  Hopkins-Cole  Reaction.^ — Place  1-2  c.c.  of  egg  albumin  solu- 
tion and  3  c.c.  of  glyoxylic  acid,  CHO.COOH  +  H^O  or  CH(OH)2- 
COOH,  solution  (Hopkins-Cole  reagent^)  in  a  test-tube  and  mix  thor- 
oughly. In  a  second  tube  place  5  c.c.  of  concentrated  sulphuric  acid. 
Incline  the  tube  containing  the  sulphuric  acid  and  by  means  of  a  pipette 
allow  the  albumin-glyoxylic  acid  solution  to  flow  carefully  down  the  side. 
When  stratified  in  this  manner  a  reddish-violet  color  forms  at  the  zone  of 
contact  of  the  two  fluids.  This  color  is  due  to  the  presence  of  the  trypto- 
phane group.  Gelatin  does  not  respond  to  this  test.  For  formula  for 
tryptophane  see  page  82. 

Benedict^  has  recently  suggested  a  new  reagent  for  use  in  carrying  out 
the  Hopkins-Cole  reaction.* 

5.  Biuret  Test. — To  2-3  c.c.  of  egg  albumin  solution  in  a  test-tube 
add  an  equal  volume  of  concentrated  potassium  hydroxide  solution,  mix 
thoroughly,  and  add  slowly  a  very  dilute  (2-5  drops  in  a  test-tube  of 
water)  copper  sulphate  solution  until  a  purplish-violet  or  pinkish-violet 
color  is  produced.  The  depth  of  the  color  depends  upon  the  nature  of  the 
protein;  proteoses,  and  peptones  giving  a  decided  pink,  while  the  color 
produced  with  gelatin  is  not  far  removed  from  a  blue.  This  reaction  is 
given  by  those  substances  which  contain  two  amino  groups  in  their  molecule, 
these  groups  either  being  joined  directly  together  or  through  a  single 
atom  of  nitrogen  or  carbon.  The  amino  groups  mentioned  must  either 
be  two  CONH2  groups  or  one  CONH,  group  and  one  CSNH2,  C(NH)- 
NHj  or  CH2NH2  group.  It  follows  from  this  fact  that  substances  which 
are  non-protein  in  character  but  which  contain  the  necessary  groups  will 
respond  to  the  biuret  test.  As  examples  of  such  substances  may  be  cited 
oxamide, 

'  Hopkins  and  Cole:    Journal  of  Physiology,  27,  418,  1902. 

^  Hopkins-Cole  reagent  is  prepared  as  follows:  To  one  liter  of  a  saturated  solution 
of  oxalic  acid  add  60  grams  of  sodium  amalgam  and  allow  the  mixture  to  stand  until  the 
evolution  of  gas  ceases.     Filter  and  dilute  with  2-3  volumes  of  water. 

^  Beneriict:    Journal  uf  Biological  Chemistry,  6,  51,  1909. 

*  Benedict's  modified  Hopkins-Cole  reagent  is  prepared  as  follows:  Ten  grams  of 
powdered  magnesium  are  placed  in  a  large  Erlerimeyer  flask  and  shaken  up  with  enough  dis- 
tilled water  to  liberally  cover  the  magnesium.  Two  hundred  and  fifty  c.c.  of  a  cold,  saturated 
solution  of  oxalic  acid  is  now  added  slowly.  The  reaction  proceeds  verj'  rapidly  and  with  the 
liberation  of  much  heat,  so  that  the  flask  should  be  cooled  under  running  water  during  the 
addition  of  the  acid.  The  contents  of  the  flask  are  shaken  after  the  addition  of  the  last 
jxjrtion  of  the  acid  and  then  poured  upon  a  Alter,  to  remove  the  insoluble  magnesium  oxalate. 
A  little  wash  water  is  pfjured  through  the  Alter,  the  filtrate  a(  idified  with  acetic  acid  to  prevent 
the  partial  precipitation  of  the  magnesium  on  long  standing,  and  made  up  to  a  liter  with 
distilled  water.     This  solution  contains  only  the  magnesium  salt  of  glyoxylic  acid. 


PROTEINS,  99 

CONH, 

I 

CONH^ 
and  biuret, 

CONH2 

\ 
NH. 

CONH, 

The  test  derives  its  name  from  the  fact  that  this  latter  substance  which 
is  formed  on  heating  urea  to  180°  C.  (see  page  286),  will  respond  to  the 
test.  Protein  material  responds  positively  since  there  are  two  CONH, 
groups  in  the  protein  molecule. 

According  to  Schiff  the  end-reaction  of  the  biuret  test  is  dependent 
upon  the  formation  of  a  copper-potassium-biuret  compound  (cupri-potas- 
sium  biuret  or  biuret  potassium  cupric  hydroxide).  This  substance  was 
obtained  by  Schiflf  in  the  form  of  long  red  needles.  It  has  the  following 
formula : 

OH  OH 

I  ■    ! 

CO  -NH, Cu NH,CO 

\  / 

NH  HN 

/  \ 

CO  NH,— K       K— NH,CO 

I      "  I      " 

OH  OH 

6.  Gies's  Biuret  Reagent.^ — Gies  has  recently  devised  a  reagent  for 
use  in  the  biuret  test.  This  reagent  consists  of  10  per  cent  KOH  solution, 
to  which  enough  3  per  cent  CuSO^  solution  has  been  added  to  impart  a 
slight  though  distinct  blue  color  to  the  clear  liquid.  The  CuSO^  should 
be  added  drop  by  drop  with  thorough  shaking  after  each  addition.  This 
reagent  is  of  material  assistance  in  performing  the  biuret  test. 

7.  Biuret  Paper  of  Kantor  and  Gies. — According  to  Kantorand  Gies^ 
when  filter  paper  is  immersed  in  the  above  reagent  and  subsequently 
dried  it  forms  a  very  satisfactory  "biuret  paper"  which  may  be  used  in  a 
manner  analogous  to  indicator  papers.  Moist  papers  may  be  used  in  the 
examination  of  powders  which  are  neutral  or  alkaline  in  reaction.  In 
preparing  the  "biuret  paper"  if  the  filter  paper  is  left  for  a  suflficient  length 
of  time  in  the  reagent  all  traces  of  the  copper  sulphate  will  be  removed 
from  the  solution. 

'  Gies:    Proceedings  of  Society  of  Biological  Chemists,  Journal  of  Biological  Chemistry, 
7,  60,  1910. 

-  Kantor  and  Gies:   Proc.  Soc.  Biol.  Chem.,  p.  ii,  1910. 


lOO  PHYSIOLOGICAL    CHEMISTRY. 

8.  Posner's  Modification  of  the  Biuret  Test. — This  test  is  par- 
ticularly satisfactory  for  use  on  dilute  protein  solutions,  and  is  carried  out 
as  follows:  To  some  dilute  egg  albumin  in  a  test-tube  add  one-half  its 
volume  of  potassium  hydroxide  solution.  Now  hold  the  tube  in  an 
inclined  position  and  allow  some  very  dilute  copper  sulphate  solution, 
made  as  suggested  on  page  98  (5),  to  flow  down  the  side,  being  especially 
careful  to  prevent  the  fluids  from  mixing.  At  the  juncture  of  the  two 
solutions  the  typical  end-reaction  of  the  biuret  test  should  appear  as  a 
colored  zone  (see  Biuret  Test,  page  98). 

9.  Testing  Colored  Solutions  by  Biuret  Test. — If  the  color  of  the 
solution  is  such  as  to  interfere  with  the  end-reaction  of  the  biuret  test 
proceed  as  follows:  Make  the  solution  strongly  alkaline  with  potassium 
hydroxide  and  add  a  solution  of  copper  sulphate.  Shake  up  the  mixture 
with  alcohol  and  if  protein  is  present  the  alcohol  vdW  assume  the  typical 
biuret  coloration.  This  procedure  is  not  applicable  in  case  the  pigment 
of  the  original  solution  is  soluble  in  alcohol.  Excess  of  the  copper  salt 
need  not  be  avoided  in  this  test. 

ID.  Liebermann's  Reaction. — Add  about  10  drops  of  concentrated 
egg  albumin  solution  (or  a  little  dry  egg  albumin)  to  about  5  c.c.  of  con- 
centrated HCl  in  a  test-tube.  Boil  the  mixture  until  a  pinkish-violet 
color  results.  This  color  was  originally  supposed  to  indicate  the  presence 
of  a  carbohydrate  group  in  the  protein  molecule,  the  furfurol  formed 
through  the  action  of  the  acid  upon  the  protein  reacting  with  the  hydroxy- 
phenyl  group  of  the  protein  producing  the  pinkish-\'iolet  color.  It  is  now 
considered  uncertain  whether  the  carbohydrate  group  enters  into  the 
reaction.  Cole  has  called  attention  to  the  fact  that  a  blue  color  results  if 
protein  material  which  has  been  boiled  ^ith  alcohol  and  subsequently 
washed  with  ether  be  used  in  making  the  test.  He  believes  the  blue  color 
to  be  due  to  an  interaction  between  the  glyoxylic  acid,  which  was  present 
as  an  impurity  in  the  ether  used  in  washing  the  protein,  and  the  trypto- 
phane group  of  the  protein  molecule  which  was  split  off  through  the  action 
of  the  acid. 

II.  Acree-Rosenheim  Formaldehyde  Reaction. — Add  a  few  drops 
of  a  dilute  (1:5000)  solution  of  formaldehyde  to  2-3  c.c.  of  egg  albumin 
solution  in  a  test-tube.  Mix  thoroughly  and  after  2-3  minutes  carefully 
introduce  a  little  concentrated  sulphuric  acid  into  the  tube  rn  such  a 
manner  that  the  two  solutions  do  not  mix.  A  \'iolet  zone  will  be  observed 
at  the  point  of  juncture  of  the  two  solutions  especially  if  the  mixture  is 
slightly  agitated.  This  color  probably  results  through  the  union  of  the 
protein  and  the  formaldehyde.  If  the  sulphuric  acid  is  added  to  the 
protein  before  the  formaldehyde  is  added  the  typical  end-reaction  is  not 
obtained.      So    far   as   is   known   this  is   a  specific   test   for  proteins. 


PROTEINS,  lOI 

The  reaction  cannot  be  applied  satisfactorily  with  concentrated  formal- 
dehyde. 

Rosenheim  claims  the  reaction  is  due  to  the  presence  of  oxidizing 
material  in  the  sulphuric  acid  and  that  when  pure  sulphuric  acid  is  used 
no  reaction  is  obtained.  He  advises  the  use  of  a  slight  amount  of  an 
oxidizing  agent,  e.  g.,  ferric  chloride  or  potassium  nitrate  (0.005  K^am  per 
100  c.c.  of  sulphuric  acid)  in  order  to  facilitate  the  reaction.  Rosenheim 
further  states  tTiat  proteins  respond  to  the  formaldehyde  reaction  because 
of  the  presence  of  the  tryptophane  group,  a  statement  which  Acree  does  not 
accept  as  proven. 

12.  Bardach's  Reaction/ — This  is  one  of  the  most  recent  tests  which 
have  been  described  for  the  detection  of  protein  material.  The  test 
depends  upon  the  property  possessed  by  protein  substances  of  preventing 
the  formation  of  typical  iodoform  crystals  through  the  interaction  of  an 
alkaline  acetone  solution  with  iodopotassium  iodide.  Instead  of  the 
typical  hexagonal  plates  or  stellar  formations  of  iodoform  there  are  pro- 
duced, under  the  conditions  of  the  test,  fine  yellow  needles  which  are 
apparently  some  iodine  compound  other  than  iodoform.  The  technic 
of  the  test  is  as  follows:  Place  about  5  c.c.  of  the  protein  solution^  under 
examination  in  a  test-tube,  add  2-3  drops  of  a  0.5  per  cent  solution  of 
acetone  and  sufficient  Lugol's  solution^  to  supply  a  moderate  excess  of 
iodine  and  produce  a  red-brown  coloration.  (The  amount  of  Lugol's 
solution  necessary  will  depend  upon  the  content  of  protein,  sugar,  and 
other  iodine-reacting  substances  in  the  solution  under  examination  and 
may  vary  from  one  drop  to  several  cubic  centimeters.)  Add  an  excess 
(ordinarily  about  3  c.c.)  of  concentrated  ammonium  hydroxide  and 
thoroughly  mix  the  solution.  Place  the  tube  in  the  test-tube  rack,  ex- 
amine the  contents  at  intervals  of  five  minutes,  and  when  it  is  evident  that 
crystals  have  formed,  place  a  drop  of  the  mixture  upon  a  microscopic 
slide,  put  a  coverglass  in  position,  and  examine  the  mixture  under  the 
microscope.  The  formation  of  canary  yellow  crystals  indicates  the  pres- 
ence of  protein  material  in  the  solution  examined.  The  crystals  are 
ordinarily  needle-like  in  appearance  and  show  a  tendency  to  assume 
rosette  or  bundle-like  formations,  but  under  certain  conditions  they  may 
show  knobbed  (nail-like)  and  branching  variations. 

If  a  moderate  excess  of  iodine  is  used  in  making  the  test,  a  black  pre- 
cipitate of  iodonitro  compounds  is  at  once  formed  upon  the  addition  of 
the  ammonium  hydroxide,  and  yellow  needles  are  subsequently  deposited 
upon  it.     In  case  just  the  proper  amount  of  iodine  is  used,  the  solution 

*  Bardach:  Zeitschrift  fur  Physiologische  Chemie,  54,  355,  iqoS;  also  Seaman  and 
Gies:    Proceedings  of  the  Society  for  Experimental  Biology  and  Medicine,  5,  125,  1908. 

*  The  solution  should  not  contain  more  than  5  per  cent  of  protein  material. 

'  Dissolve  4  grams  of  iodine  and  6  grams  of  potassium  iodide  in  100  c.c.  of  distilled  water. 


I02  PHYSIOLOGICAL    CHEMISTRY. 

soon  assumes  a  yellow  color  and  the  black  precipitate  formed  upon  the 
addition  of  the  ammonium  hydroxide  is  gradually  transformed  more  or 
less  completely  into  theyellow  crystals.  In  either  case  the  needles  ordinarily 
form  within  an  hour,  and  frequently  in  a  much  shorter  time.  If  too  great 
an  excess  of  iodine  is  employed  the  heavy  black  precipitate  may  obscure 
or  even  prevent  the  reaction.  The  presence  of  insufficient  iodine  or 
excess  protein  may  likewise  prevent  the  reaction.  In  tests  in  which  a 
concentrated  protein  solution  and  an  excess  of  iodine  are  used,  the  addi- 
tion of  ammonium  hydroxide  immediately  produces  a  grayish-green 
precipitate.  In  such  instances,  if  the  proportions  are  favorable,  and  the 
mixture  be  stirred  with  a  glass  rod  for  a  few  minutes,  the  precipitate  is 
gradually  transformed  into  the  crystals  before  mentioned. 

It  is  probable  that  all  soluble  proteins  will  respond  to  Bardach's 
reaction,  but  the  relative  delicacy  of  the  reaction  as  well  as  the  value  of  the 
test  as  compared  with  other  protein  tests  remain  to  be  determined.  The 
only  disturbing  factor  noted  thus  far  is  the  presence  of  earthy  phosphates 
in  the  solution  under  examination. 

PRECIPITATION  REACTIONS  AND  OTHER  PROTEIN  TESTS. 

There  are  three  forms  in  which  proteins  may  be  precipitated,  i.  e., 
unaltered,  as  an  albitminate,  and  as  an  insoluble  salt.  An  instance  of  the 
precipitation  in  a  native  or  unaltered  condition  is  seen  in  the  so-called 
salting-out  experiments.  Various  salts,  notably  (NHJ2SO4,  ZnSO^, 
MgSO^,  NajSO^  and  NaCl  possess  the  power  when  added  in  solid  form  to 
certain  definite  protein  solutions,  of  rendering  the  menstruum  incapable  of 
holding  the  protein  in  solution,  thereby  causing  the  protein  to  be  precipi- 
tated or  salted-out  to  use  the  common  term.  Mineral  acids  and  alcohol 
also  precipitate  proteins  unaltered.  In  the  case  of  concentrated  acids 
the  protein  is  dissolved  in  the  presence  of  an  excess  of  acid  with  the 
formation  of  a  protein  salt.  Proteins  are  precipitated  as  albuminates 
(protein  salts)  when  treated  with  certain  metallic  salts,  and  precipitated  as 
insoluble  salts  when  weak  organic  acids  such  as  certain  of  the  alkaloidal 
reagents  are  added  to  their  solutions. 

If  certain  acids  (picric,  tungstic,  phosphomolybdic,  tannic,  or  chromic) 
be  added  to  a  neutral  albumin  solution  a  precipitate  of  a  protein  salt 
occurs.  If,  however,  the  salts  of  these  acids  be  added  no  precipitate 
occurs.  The  addition  of  a  small  amount  of  acid,  as  acetic  acid,  to  such  a 
solution  will  cause  a  precipitate  to  form.' 

The  effect  of  the  addition  of  the  salts  of  the  heavy  metals  is  in  the  first 
instance  to  cause  a  precipitation  of  the  protein.     In  many  cases,  however, 

'  Mathews:    Amer.  Jour,  of  Physiology,  1,  445,  1898. 


PROTEINS.  103 

the  addition  of  an  excess  of  such  saUs  causes  the  solution  of  the  precipitate 
while  a  further  excess  may  cause  a  reprecipitation.  The  precipitate  which 
is  first  formed  in  a  protein  solution  by  the  addition  of  the  salts  of  the  heavy 
metals  may  be  redissolved  not  only  by  an  excess  of  such  salts  but  by  an 
excess  of  protein  as  well/ 

It  is  generally  stated  that  globulins  are  ])recij)itated  from  their  solutions 
upon  half  saturation  with  ammonium  suli)hate  and  that  albumins  are 
precipitated  upon  complete  saturation  by  this  salt.  Comparatively  few 
exceptions  were  found  to  this  rule  until  proteins  of  vegetable  origin  came 
to  be  more  extensively  studied.  These  studies,  furthered  especially  by 
Osborne  and  associates,  have  demonstrated  very  clearly  that  the  char- 
acterization of  a  globulin  as  a  protein  which  is  precipitated  by  half 
saturation  with  ammonium  sulphate,  can  no  longer  hold.  Certain  vege- 
table globulins  have  been  isolated  which  are  not  precipitated  by  this  salt 
until  a  concentration  is  reached  greater  than  that  secured  by  half-saturation. 
As  an  example  of  an  albumin  which  does  not  conform  to  the  definition  of 
an  albumin  as  regards  its  precipitation  by  ammonium  sulphate,  may  be 
mentioned  the  leucosin  of  the  wheat  germ  which  is  precipitated  from  its 
solution  upon  /;tz//-saturation  with  ammonium  sulphate.  The  limits  of 
precipitation  by  ammonium  sulphate,  therefore,  do  not  furnish  a  suffi- 
ciently accurate  basis  for  the  differentiation  of  globulins  from  albumins. 
It  has  further  been  determined  that  a  given  protein  which  is  precipitable 
by  ammonium  sulphate  cannot  be  "  salted-out "  by  the  same  concentration 
of  the  salt  under  all  conditions. 

Experiments. 

1.  Influence  of  Concentrated  Mineral  Acids,  Alkalis  and  Organic 

Acids. — Prepare  five  test-tubes  each  containing  5  c.c.  of  concentrated  egg 
albumin  solution.  To  the  first  add  concentrated  HjSO^,  drop  by  drop, 
until  an  excess  of  the  acid  has  been  added.  Note  any  changes  which  may 
occur  in  the  solution.  Allow  the  tube  to  stand  for  24  hours  and  at  the 
end  of  that  period  observe  any  alteration  which  may  have  taken  place. 
Heat  the  tube  and  note  any  further  change  which  may  occur.  Repeat  the 
experiment  in  the  four  remaining  tubes  with  concentrated  hydrochloric 
acid,  concentrated  nitric  acid,  concentrated  potassium  hydroxide  and 
acetic  acid.  How  do  strong  mineral  acids,  strong  alkalis,  and  strong 
organic  acids  differ  in  their  action  toward  protein  solutions? 

2.  Precipitation  by  Metallic  Salts. — Prepare  four  tubes  each  con- 
taining 2-3  c.c.  of  dilute  egg  albumin  solution.     To  the  first  add  mercuric 

*  Pauli:  Hofmeister's  Beitrage,  6,  233,  1904-5.  Robertson:  Ergebnisse  tier  Physiologic, 
10,  290,  19T0. 


I04  PHYSIOLOGICAL    CHEMISTRY. 

chloride,  drop  by  drop,  until  an  excess  of  the  reagent  has  been  added,  noting 
any  changes  which  may  occur.  Repeat  the  experiment  with  lead  acetate, 
silver  nitrate,  copper  sulphate,  ferric  chloride,  and  barium  chloride. 

Egg  albumin  is  used  as  an  antidote  for  lead  or  mercury  poisoning. 
Why? 

3.  Precipitation  by  Alkaloidal  Reagents. — ^Prepare  six  tubes  each 
containing  2-3  c.c.  of  dilute  egg  albumin  solution.  To  the  first  add 
picric  acid  drop  by  drop  until  an  excess  of  the  reagent  has  been  added, 
noting  any  changes  which  may  occur.  Repeat  the  experiment  with 
trichloracetic  acid,  tannic  acid,  phosphotungstic  acid,  phosphomolybdic  acid, 
and  potassio-mercuric  iodide.  Acidify  with  hydrochloric  acid  before  testing 
with  the  three  last  reagents. 

4.  Heller's  Ring  Test. — ^Place  5  c.c.  of  concentrated  nitric  acid  in  a 
test-tube,  incline  the  tube,  and  by  means  of  a  pipette  allow  the  dilute 
albumin  solution  to  flow  slowly  down  the  side.  The  liquids  should  stratify 
with  the  formation  of  a  white  zone  of  precipitated  albumin  at  the  point  of 
juncture.     This  is  a  very  delicate  test  and  is  further  discussed  on  p.  333. 

An  apparatus  called  the  albumoscope  or  horismascope  has  been  devised 
for  use  in  the  tests  of  this  character  and  has  met  with  considerable  favor. 
The  method  of  using  the  albumoscope  is  described  below. 

Use  of  the  Albumoscope. — This  instrument  is  intended  to  facilitate 
the  making  of  "ring"  tests  such  as  Heller's  and  Roberts'.  In  making  a 
test  about  5  c.c.  of  the  solution  under  examination  is  first  introduced  into 
the  apparatus  through  the  larger  arm  and  the  reagent  used  in  the  particu- 
lar test  is  then  introduced  through  the  capillary  arm  and  allowed  to  flow 
down  underneath  the  solution  under  examination.  If  a  reasonable 
amount  of  care  is  taken  there  is  no  possibility  of  mixing  the  two  solutions 
and  a  definitely  defined  white  "ring"  is  easily  obtained  at  the  zone  of 
contact. 

5.  Roberts'  Ring  Test. — Place  5  c.c.  of  Roberts'  reagent^  in  a  test- 
tube,  incline  the  tube,  and  by  means  of  a  pipette  allow  the  albumin  solu- 
tion to  flow  slowly  down  the  side.  The  liquids  should  stratify  with  the 
formation  of  a  white  zone  of  precipitated  albumin  at  the  point  of  juncture. 
This  test  is  a  modification  of  Heller's  ring  test  and  is  rather  more  satis- 
factory. The  albumoscope  may  also  be  used  in  making  this  test.  (See 
page  334. j 

6.  Spiegler's  Ring  Test. — Place  5  c.c.  of  Spiegler's  reagent^  in  a  test- 

*  Roberts'  reagent  is  composed  of  i  volume  of  concentrated  HNO3  and  5  volumes  of  a 
saturated  solution  of  MgSO^. 

*  Spiegler's  reagent  has  the  following  composition: 

Tartaric  acid 20  grams. 

Mercuric  chloride 40  grams. 

Glycerol 100  grams. 

Distilled  water 1000  grams. 


PROTEINS.  TO 


tube,  incline  the  tube,  and  by  means  of  a  pipette  allow  5  c.c.  of  albumin 
solution,  acidified  with  acetic  acid,  to  flow  slowly  down  the  side.  A 
white  zone  will  form  at  the  point  of  contact.  This  is  an  exceedingly 
delicate  test,  in  fact  too  delicate  for  ordinary  clinical  purposes,  since  it 
serves  to  detect  albumin  when  present  in  the  merest  trace  (i  : 250,000). 
This  test  is  further  discussed  on  page  335. 

7.  Jolles'  Reaction. — Shake  5  c.c.  of  albumin  solution  with  i  c.c.  of 
30  per  cent  acetic  acid  and  4  c.c.  of  Jolles'  reagent^  in  a  test-tube.  A 
white  precipitate  of  albumin  should  form.  Care  should  be  taken  to  use 
the  correct  amount  of  acetic  acid.     For  further  discussion  of  the  test  see 

page  335- 

8.  Tanret's  Test. — To  5  c.c.  of  albumin  solution  in  a  test-tube  add 
Tanret's  reagent,^  drop  by  drop,  until  a  turbidity  or  precipitate  forms. 
This  is  an  exceedingly  deHcate  test.  Sometimes  the  albumin  solution  is 
stratified  upon  the  reagent  as  in  Heller's  or  Roberts'  ring  tests.  In  urine 
examination  it  is  claimed  by  Repiton  that  the  presence  of  urates  lowers 
the  delicacy  of  the  test.  Tanret  has,  however,  very  recently  made  a  state- 
ment to  the  effect  that  the  removal  of  urates  is  not  necessary  inasmuch  as 
the  urate  precipitate  will  disappear  on  warming  and  the  albumin  precipi- 
tate will  not.  He  says,  however,  that  mucin  interferes  with  the  delicacy 
of  his  test  and  should  be  removed  by  acidification  with  acetic  acid  and 
filtration    before   testing   for   albumin. 

9.  Sodium  Chloride  and  Acetic  Acid  Test. — Mix  2  volumes  of 
albumin  solution  and  i  volume  of  a  saturated  solution  of  sodium  chloride 
in  a  test-tube,  acidify  with  acetic  acid,  and  heat  to  boiling.  The  pro- 
duction of  a  cloudiness  or  the  formation  of  a  precipitate  indicates  the 
presence  of  albumin. 

10.  Potassium  Iodide  Test. — Stratify  a  dilute  albumin  solution 
upon  a  solution  of  potassium  iodide  made  slightly  acid  with  acetic  acid. 
In  the  presence  of  0.01-0.02  per  cent  of  albumin  a  white  ring  forms 
immediately.  If  the  test  be  allowed  to  stand  two  minutes  after  the 
stratification  it  will  serve  to  detect  0.005  P^r  cent  of  albumin. 

11.  Acetic  Acid  and  Potassium  Ferrocyanide  Test. — To  5  c.c. 
of  dilute  egg  albumin  solution  in  a  test-tube  add  5-10  drops  of  acetic 
acid.  Mix  well,  and  add  potassium  ferrocyanide,  drop  by  drop,  until 
a  precipitate  forms.     This  test  is  very  delicate. 

'  Jolles'  reagent  has  the  following  composition: 

Succinic  acid 40  grams. 

Mercuric  chloride 20  grams. 

Sodium  chloride 20  grams. 

Distilled  water 1000  grams. 

^Tanret's  reagent  is  prepared  as  follows:  Dissolve  1.35  gram  of  mercuric  chloride  in  25 
c.c.  of  water,  add  to  this  solution  3.32  grams  of  potassium  iodide  dissolved  in  25  c.c.  of  water, 
then  make  the  total  solution  up  to  60  c.c.  with  water  and  add  20  c.c.  of  glacial  acetic  acid  to 
the  combined  solutions. 


Io6  PHYSIOLOGICAL    CHEMISTRY. 

Schmiedl  claims  that  a  precipitate  of  Fe(Cn)gK2Zn  or  Fe(Cn)Q- 
Zn^,  is  formed  when  solutions  containing  zinc  are  subjected  to  this  test, 
and  that  this  precipitate  resembles  the  precipitate  secured  with  protein 
solutions.  In  the  case  of  human  urine  a  reaction  was  obtained  when 
0.000022  gram  of  zinc  per  cubic  centimeter  was  present.  Schmiedl 
further  found  that  the  urine  collected  from  rabbits  housed  in  zinc-lined 
cages  possessed  a  zinc  content  which  was  sufl&cient  to  yield  a  ready  re- 
sponse to  the  test.     Zinc  is  the  only  interfering  substance  so  far  reported. 

12.  Salting-out  Experiments. — (a)  To  25  c.c.  of  egg  albumin 
solution  in  a  small  beaker  add  solid  ammonium  sulphate  to  the  point 
of  saturation,  keeping  the  temperature  of  the  solution  below  40°  C. 
Filter,  test  the  precipitate  by  Millon's  reaction  and  the  filtrate  by  the  biu- 
ret test.  What  are  your  conclusions?  (&)  Repeat  the  above  experi- 
ment making  the  saturation  with  solid  sodium  chloride.  How  does 
this  result  differ  from  the  result  of  the  saturation  with  ammonium  sul- 
phate ?  Add  2-3  drops  of  acetic  acid.  .  What  occurs  ?  All  proteins 
except  peptones  are  precipitated  by  saturating  their  solutions  with  ammo- 
nium sulphate.  Glohulins  are  the  only  proteins  precipitated  by  satu- 
rating with  sodium  chloride  (see  Globulins,  page  109),  unless  the  satu- 
rated solution  is  subsequently  acidified,  in  which  event  all  proteins  except 
peptones  are  precipitated. 

Soaps  may  be  salted-out  in  a  similar  manner  (see  p.  145). 

13.  Coagulation  or  Boiling  Test. — Heat  25  c.c.  of  dilute  egg 
albumin  solution  to  the  boiling-point  in  a  small  evaporating  dish.  The 
albumin  coagulates.  Complete  coagulation  may  be  obtained  by  acidify- 
ing the  solution  with  3-5  drops  of  acetic  acid^  at  the  boiling-point.  Test 
the  coagulum  by  Millon's  reaction.  The  acid  is  added  to  neutralize  any 
possible  alkalinity  of  the  solution,  to  dissolve  any  substances  which  are 
not  albumin  and  to  facilitate  coagulation  (see  further  discussion  on  pages 
117  and  335). 

14.  Coagulation  Temperature. — Prepare  4  test-tubes  each  con- 
taining 5  c.c.  of  neutral  egg  albumin  solution.  To  the  first  add  i  drop  of 
0.2  per  cent  hydrochloric  acid,  to  the  second  add  i  drop  of  o. 5  per  cent 
sodium  carbonate  solution,  to  the  third  add  i  drop  of  10  per  cent  sodium 
chloride  solution  and  leave  the  fourth  neutral  in  reaction.  Partly  fill  a 
beaker  of  medium  size  with  water  and  place  it  within  a  second  larger 
beaker  which  also  contains  water,  the  two  vessels  being  separated  by 
pieces  of  cork.  Fasten  the  four  test-tubes  compactly  together  by  means 
of  a  rubber  band,  lower  them  into  the  water  of  the  inner  beaker  and  sus- 
pend them,  by  means  of  a  clamp  attached  to  one  of  the  tubes,  in  such  a 

'  Nitric  acid  is  often  used  in  place  of  acetic  acid  in  this  test.  In  case  nitric  acid  is  used, 
ordinarily^i-2  drops  is  sufTu  ient. 


PROTEINS. 


Jo: 


0&  P 


Q--^ 


manner  that  the  albumin  solutions  shall  be  midway  between  the  upper 
and  lower  surfaces  of  the  water.  In  one  of  the  tubes  place  a  thermometer 
with  its  bulb  entirely  beneath  the  surface  of  the  albumin  solution  (Fig.  33). 
Gently  heat  the  water  in  the  beakers,  noting  carefully  any  changes  which 
may  occur  in  the  albumin  solutions  and  record  the  exact  tem- 
perature at  which  these  changes  occur.  The  first  appearance  of  an 
opacity  in  an  albumin  solution  indicates  the 
commencement  of  coagulation  and  the 
temperature  at  which  this  occurs  should 
be  recorded  as  the  coagulation  temperature 
for  that  particular  albumin  solution. 

What  is  the  order  in  which  the  four 
solutions  coagulate? 

Repeat  the  experiment,  adding  to  the 
first  tube  i  drop  of  acetic  acid,  to  the  second 
I  drop  of  concentrated  potassium  hydroxide 
solution,  to  the  third  2  drops  of  a  10  per 
cent  sodium  chloride  solution  and  leave 
the  fourth  neutral  as  before. 

What  is  the  order  of  coagulation  here  ? 
Why  ? 

15.  Precipitation  by  Alcohol. — Pre- 
pare 3  test-tubes  each  containing  about  10 
c.c.  of  95  per  cent  alcohol.  To  the  first 
add  one  drop  of  0.2  per  cent  hydrochloric 
acid,  to  the  second  one  drop  of  potassium 
hydroxide  solution  and  leave  the  third 
neutral  in  reaction.  Add  to  each  tube  a 
few  drops  of  egg  albumin  solution  and 
note  the  results.  What  do  you  conclude 
from  this  experiment  ?  Alcohol  precipitates 
proteins  unaltered,  but  if  allowed  to  re- 
main under  alcohol  the  protein  is  trans- 
formed. The  "fixing"  of  tissues  for  histological  examination  by  means 
of  alcohol  is  an  illustration  of  the  application  of  this  transformation  pro- 
duced by  alcohol.     It  apparently  is  a  process  of  dehydration. 

16.  Preparation  of  Powdered  Egg  Albumin. — This  may  be  pre- 
pared as  follows :  Ordinary  egg-white  finely  divided  by  means  of  scissors 
or  a  beater  is  treated  with  four  volumes  of  water  and  filtered.  The 
filtrate  is  evaporated  on  a  water-bath  at  about  50°  C.  and  the  residue 
powdered  in  a  mortar. 

17.  Tests  on  Powdered  Egg  Albumin. — With  powdered  albumin 


Fig. 


5. — Co.-VGULATiox  Temper- 
ature Apparatus. 


Io8  PHYSIOLOGICAL    CHEMISTRY. 

prepared  as  described  above  (by  yourself  or  furnished  by  the  instructor)  ^ 
try  the  following  tests: 

(a)  Solubility. 

{b)  Millon's  Reaction. 

(c)  Hopkins-Cole  Reaction. — When  used  to  detect  the  presence  of 
protein  in  solid  form  this  reaction  should  be  conducted  as  follows:  Place 
5  c.c.  of  concentrated  sulphuric  acid  in  a  test-tube  and  add  carefully, 
by  means  of  a  pipette,  3-5  c.c.  of  Hopkins-Cole  reagent.  Introduce  a 
small  amount  of  the  solid  substance  to  be  tested,  agitate  the  tube  slightly, 
and  note  that  the  suspended  pieces  assume  a  reddish-violet  color,  which 
is  the  characteristic  end-reaction  of  the  Hopkins-Cole  test;  later  the 
solution  will  also  assume  the  reddish-violet  color. 

(d)  Composition  Test. — Heat  some  of  the  powder  in  a  test-tube  in 
which  is  suspended  a  strip  of  moistened  red  litmus  paper  and  across  the 
mouth  of  which  is  placed  a  piece  of  filter  paper  moistened  with  lead 
acetate  solution.  As  the  powder  is  heated  it  chars,  indicating  the  presence 
of  carbon;  the  fumes  of  ammonia  are  evolved,  turning  the  red  litmus 
paper  blue  and  indicating  the  presence  of  nitrogen  and  hydrogen;  the 
lead  acetate  paper  is  blackened,  indicating  the  presence  of  sulphur^ 
and  the  deposition  of  moisture  on  the  side  of  the  tube  indicates  the 
presence  of  hydrogen. 

(e)  Immerse  a  dry  test-tube  containing  a  little  powdered  egg  albumin 
in  boiling  water  for  a  few  moments.  Remove  and  test  the  solubility  of  the 
albumin  according  to  the  directions  given  under  (a)  above.  It  is  still 
soluble.  Why  has  it  not  been  coagulated?  Repeat  the  above  experi- 
ments with  powdered  serum  albumin  and  see  how  the  results  compare 
with  those  just  obtained. 

SULPHUR  IN  PROTEIN. 

Sulphur  is  believed  to  be  present  in  two  different  forms  in  the  pro- 
tein molecule.  The  first  form,  which  is  present  in  greatest  amount^ 
is  that  loosely  combined  with  carbon  and  hydrogen.  Sulphur  in  this 
form  is  variously  termed  unoxidized,  loosely  combined,  mercaptan,  and 
lead-blackening  sulphur.  The  second  form  is  combined  in  a  more  stable 
manner  with  carbon  and  oxygen  and  is  known  as  oxidized  or  acid  sulphur. 
The  protamines  are  the  only  class  of  sulphur-free  proteins. 

Tests  for  Sulphur. 

I.  Tests  for  Loosely  Combined  Sulphur. — (a)  To  equal  volumes  of 
KOH  and  egg  albumin  solutions  in  a  test-tube  add  1-2  drops  of  lead 
acetate  solution  and  boil  the  mixture.     Loosely  combined  sulphur  is 


PROIKINS.  109 

indicated  by  a  darkening  of  the  solution,  the  color  deepening  into  a 
black  if  sufl'icicnt  sulphur  is  present.  Add  hydrochloric  acid  and  note 
the  characteristic  odor  evolved  from  the  solution.  Write  the  reactions 
for  this  test,  (b)  Place  equal  volumes  of  KOH  and  egg  albumin  solu- 
tions in  a  test-tube  and  boil  the  mixture  vigorously.  Cool,  make  acid 
with  glacial  acetic  acid. and  add  1-2  drops  of  lead  acetate.  A  darken- 
ing indicates  the  presence  of  loosely  combined  sulphur. 

2.  Test  for  Total  Sulphur  (Loosely  Combined  and  Oxidized). — 
Place  the  substance  to  be  examined  (powdered  egg  albumin)  in  a  small 
porcelain  crucible,  add  a  suitable  amount  of  solid  fusion  mixture  (potas- 
sium hydroxide  and  potassium  nitrate  mixed  in  the  proportion  5:1)  and 
heat  carefully  until  a  colorless  mixture  results.  (Sodium  peroxide  may 
be  used  in  place  of  this  fusion  mixture  if  desired.)  Cool,  dissolve  the  cake 
in  a  httle  warm  water  and  filter.  Acidify  the  filtrate  with  hydrochloric 
acid,  heat  it  to  the  boiling-point  and  add  a  small  amount  of  barium 
chloride  solution.  A  white  precipitate  forms  if  sulphur  is  present. 
What  is  this  precipitate  ? 

GLOBULINS. 

Globulins  are  simple  proteins  especially  predominant  in  the  vege- 
table kingdom.  They  are  closely  related  to  the  albumins  and  in  com- 
mon with  them  give  all  the  ordinary  protein  tests.  Globulins  differ 
from  the  albumins  in  being  insoluble  in  pure  (salt-free)  water.  They 
are,  however,  soluble  in  neutral  solutions  of  salts  of  strong  bases  with 
strong  acids.  Most  globulins  are  precipitated  from  their  solutions  by 
saturation  with  solid  sodium  chloride  or  magnesium  sulphate.  As  a 
class  they  are  much  less  stable  than  the  albumins,  a  fact  shown  by  the 
increasing  difficulty  with  which  a  globulin  dissolves  during  the  course  of 
successive  reprecipitations. 

We  have  used  an  albumin  of  animal  origin  (egg  albumin)  for  all 
the  protein  tests  thus  far,  whereas  the  globulin  to  be  studied  will  be 
prepared  from  a  vegetable  source.  There  being  no  essential  difference 
between  animal  and  vegetable  proteins,  the  vegetable  globulin  we  shall 
study  may  be  taken  as  a  true  type  of  all  globulins,  both  animal  and 
vegetable. 

Experiments  on  Globulin. 

Preparation  of  the  Globulin.— Extract  20-30  grams  (a  handful) 
of  crushed  hemp  seed  with  a  5  per  cent  solution  of  sodium  chloride  for 
one-half  hour  at  60°  C.  Filter  while  hot  through  a  paper  moistened 
with  5  per  cent  sodium  chloride  solution.  Place  the  filtrate  in  the  water- 
bath  at  60°  C.  and  allow  it  to  stand  for  24  hours  in  order  that  the  globulin 


no  PHYSIOLOGICAL    CHEMISTRY. 

may  crystallize  slowly.  In  case  the  filtrate  is  cloudy  it  should  be  warmed 
to  60°  C.  in  order  to  produce  a  clear  solution.  The  globulin  is  soluble  in 
hot  5  per  cent  sodium  chloride  solution  and  is  thus  extracted  from  the 


Fig.  34. — Edestin. 

hemp  seed,  but  upon  cooling  this  solution  much  of  the  globulin  separates  in 
crystalline  form.  This  particular  globulin  is  called  edestin.  It  crystal- 
lizes in  several  different  forms,  chiefly  octahedra  (see  Fig.  34,  above). 
(The  crystalline  form  of  excelsin,  a  protein  obtained  from  the  Brazil  nut, 
is  shown  in  Fig.  35,  below.     This  vegetable  protein  crystallizes  in  the 


Fig.  35. — Excelsin,  the  Protein  or  the  Brazil  Nut. 
(Drawn  from  crystals  furnished  by  Dr.  Thomas  B.  Osborne,  New  Haven,  Conn.) 

form  of  hexagonal  plates.)  Filter  oflf  the  edestin  and  make  the  following 
tests  on  the  crystalline  body  and  on  the  filtrate  which  still  contains  some 
of  the  extracted  globulin. 


PROTEIN'S.  1 1 1 

Tests  on  Crystallized  Edestin. — (i)  Microscopical  examination 
(see  Fig.  34,  p.  no). 

(2)  Solubility. — Try  the  solubility  in  the  ordinary  solvents  (see  page 
27).  Keep  these  solubilities  in  mind  for  comparison  with  those  of 
edestan,  to  be  made  later  (see  page  115). 

(3)  Mil  Ion's  Reaction. 

(4)  Coagulation  Test. — Place  a  small  amount  of  the  globulin  in  a 
test-tube,  add  "a  little  .water  and  boil.  Now  add  dilute  hydrochloric 
acid  and  note  that  the  protein  no  longer  dissolves.     It  has  been  coagulated. 

(5)  Dissolve  the  remainder  of  the  edestin  in  0.2  per  cent  hydro- 
chloric acid  and  preserve  this  acid  solution  for  use  in  the  experiments 
on  proteans  (see  page  115). 

Tests  on  Edestin  Filtrate. — (i)  Influence  of  Protein  Precipi- 
tants. — Try  a  few  protein  precipitants  such  as  nitric  acid,  tannic  acid, 
picric  acid,  and  mercuric  chloride. 

(2)  Biuret  Test. 

(3)  Coagulatian  Test. — Boil  some  of  the  filtrate  in  a  test-tube.  What 
happens  ? 

(4)  Saturation  with  Sodium  Chloride. — Saturate  some  of  the  filtrate 
with  solid  sodium  chloride.  How  does  this  result  differ  from  that  ob- 
tained upon  saturating  egg  albumin  solution  with  solid  sodium  chloride ':! 

(5)  Precipitation  by  Dilution. — Dilute  some  of  the  filtrate  with  ia-15 
volumes  of  water.     Why  does  the  globulin  precipitate  ? 

Glutelins. 

It  has  been  repeatedly  shown,  particularly  by  Osborne,  that  after 
extracting  the  seeds  of  cereals  with  water,  neutral  salt  solution,  and 
strong  alcohol,  there  still  remains  a  residue  which  contains  protein 
material  which  may  be  extracted  by  very  dilute  acid  or  alkali.  These 
proteins  which  are  insoluble  in  all  neutral  solvents,  but  readily  soluble 
in  very  dilute  acids  and  alkalis  are  called  glutelins.  The  only  member 
of  the  group  which  has  yet  received  a  name,  is  the  glutenin  of  wheat, 
a  protein  which  constitutes  nearly  50  per  cent  of  the  gluten.  It  is  not 
definitely  known  whether  glutelins  occur  as  constituents  of  all  seeds. 

Prolamins  (Alcohol-soluble  Proteins). 

The  term  prolamin  has  been  proposed  by  Osborne,  for  the  group  of 
proteins  formerly  termed  "alcohol-soluble  proteins."  The  name  is 
very  appropriate  inasmuch  as  these  proteins  yield,  upon  hydrolysis, 
especially  large  amounts  of  proline  and  ammania.  The  prolamins  are 
simple  proteins  which  are  insoluble  in  water,  absolute  alcohol  and  other 


112  PHYSIOLOGICAL    CHEMISTRY. 

neutral  solvents,  but  are  soluble  in  70  to  80  per  cent  alcohol  and  in  dilute 
acids  and  alkalis.  They  occur  widely  distributed,  particularly  in  the 
vegetable  kingdom.  The  only  prolamins  yet  described  are  the  zein  of 
maize,  the  hordein  of  barley,  the  gliadin  of  wheat  and  rye,  and  the  hynin 
of  malt.  They  yield  relatively  large  amounts  of  glutamic  acid  on  hydroly- 
sis but  no  lysin.  The  largest  percentage  of  glutamic  acid  (43.66  per  cent) 
ever  obtained  as  a  decomposition  product  of  a  protein  substance  has 
very  recently  been  obtained  by  Osborne  &  Guest  from  the  hydrolysis  of 
the  prolamin  gliadin.'^  This  yield  of  glutamic  acid  is  also  the  largest 
amount  of  any  single  decomposition  product  yet  obtained  from  any 
protein  except  protamines. 

Albuminoids.     (Scleroproteins.) 

The  albuminoids  yield  similar  hydrolytic  products  to  those  obtained 
from  the  other  simple  proteins  already  considered,  thus  indicating  that 
they  possess  essentially  the  same  chemical  structure.  They  differ  from 
all  other  proteins,  whether  simple,  conjugated,  or  derived,  in  that  they 
are  insoluble  in  all  neutral  solvents.  The  albuminoids  include  "the 
principal  organic  constituents  of  the  skeletal  structure  of  animals  as 
well  as  their  external  covering  and  its  appendages.  Some  of  the  principal 
albuminoids  are  keratin,  elastin,  collagen,  reticulin,  spongin,  Sind  fibroin. 
Gelatin  cannot  be  classed  as  an  albuminoid  although  it  is  a  transformation 
product  of  collagen.  The  various  albuminoids  differ  from  each  other  in 
certain  fundamental  characteristics  which  will  be  considered  in  detail 
under  Epithelial  and   Connective  Tissue    (see   Chapter  XIV,  p.   245). 

CONJUGATED  PROTEINS. 

Conjugated  proteins  consist  of  a  protein  molecule  united  to  some 
other  molecule  or  molecules  otherwise  than  as  a  salt.  We  have  glyco- 
proteins, nucleo proteins,  hcBmoglobins  (chromoproteins) ,  phospho proteins 
and  lecitho proteins  as  the  five  classes  of  conjugated  proteins. 

Glycoproteins  may  be  considered  as  compounds  of  the  protein  mole- 
cule with  a  substance  or  substances  containing  a  carbohydrate  group 
other  than  a  nucleic  acid.  The  glycoproteins  yield,  upon  decomposition, 
protein  and  carbohydrate  derivatives,  notably  glycosamine,  CHgOH.- 
(CHOH)3.CH(NH3).CHO,and  galactosamine,  GHCH^.  (CHOH)3.CH- 
(NHJ.CHO.  The  principal  glycoproteins  are  mucoids,  mucins,  and  chon- 
droproteins.  By  the  term  mucoid  we  may  in  general  designate  those  glyco- 
proteins which  occur  in  tissues,  such  as  tendomucoid  from  tendinous 

'  Up  to  this  time  the  yield  of  41.32  per  cent  obtained  by  Kleinschmitt  from  hordein  was 
the  maximum  yield. 


s. 

C. 

H. 

0. 

2-33 

48.76 

6.53 

30.60 

2.32 

47-43 

6.63 

31-40 

PROTEINS.  113 

issue  and  osseomucoid  from  bone.  The  elementary  composition  of  these 
ypical  mucoids  is  as  follows: 

N. 

Tendomucoid  • 1 1  -7  S 

Osseomucoid' 12.22 

The  term  mucins  may  be  said  in  general  to  include  those  forms  of  glyco- 
proteins which  occur  in  the  secretions  and  fluids  of  the  body.  Seroww- 
coid^  is,  however,  the  term  applied  to  the  glycoprotein  of  blood  serum. 
Chondroproteins  are  so  named  because  chondromiicoid,  the  principal 
member  of  the  group,  is  derived  from  cartilage  (chondrigen).  Amyloid,* 
which  appears  pathologically  in  the  spleen,  liver,  and  kidneys,  is  also  a 
chondroprotein. 

The  tmdeoproteins  occur  principally  in  animal  and  vegetable  cells, 
and  following  the  destruction  of  these  cells  they  are  found  in  the  fluids 
of  the  body.  These  proteins  are  discharged  into  the  tissue  fluids  by 
the  acti\-ity  or  disintegration  of  cells.  Combined  with  the  simple  pro- 
tein in  the  ncuclcoprotein  molecule  we  find  nucleic  acid,  a  body  which 
contains  phosphorus  and  which  yields  purine  bases  and  pyrimidine  bases 
{thymine,  cytosine,  and  uracil)  upon  decomposition.  The  so-called 
nucleins  are  formed  in  the  gastric  digestion  of  nucleoproteins. 

Wheeler-Johnson  Reaction  for  Uracil  and  Cytosine. — To  about 
5  c.c.  of  the  solution  under  examination  add  bromine  water  until  the  color 
is  permanent.^  In  case  the  solution  contains  only  small  quantities  of 
cytosine  or  uracil,  it  is  advisable  to  remove  the  excess  of  bromine  by 
passing  a  stream  of  air  through  the  solution.  Now  add  an  excess  of  an 
aqueous  solution  of  barium  hydroxide  and  note  the  appearance  of  a 
purple  color. 

Very  dilute  solutions  do  not  give  the  test.  Under  these  conditions  the 
solution  should  be  evaporated  to  dryness,  the  residue  dissolved  in  a 
little  bromine  water  and  the  excess  of  bromine  removed.  Then  upon 
adding  an  excess  of  barium  hydroxide  a  decided  bluish-pink  or  lavender 
color  will  appear  in  the  presence  of  as  small  an  amount  as  o.ooi  gram  of 
uracil. 

In  testing  solutions  for  cytosine,  it  is  preferable  to  warm  or  boil  the 
solution  with  bromine  water,  and  after  cooling  the  solution  to  apply  the 
test  as  suggested  above,  being  careful  to  have  a  slight  excess  of  bromine 
present  before  adding  barium  hydroxide. 

'  Chittenden  and  Gies:  Jour.  Exp.  Med,  i,  186,  1896. 

-  Hawk  and  Gies:    Amer.  Jour.  Physiol.,  5,  387,  1901. 

'  Bywaters:   Biochemische  Zeitschrfft,  15,  322,  1909. 

*  Not  to  be  confused  with  the  substance  amyloid  which  may  be  formed  from  cellulose  (see 
page  54)- 

^  Avoid  the  addition  of  a  large  excess  of  bromine  inasmuch  as  this  will  interfere  with  the 
test. 

8 


114  PHYSIOLOGICAL   CHEMISTRY. 

The  phosphoproteins  are  called  nucleo albumins  in  many  classifications 
and  are  grouped  among  the  simple  proteins.  They  are  considered  to  be 
''compounds  of  the  protein  molecule  and  some,  as  yet  undefined,  phos- 
phorus-containing substances  other  than  a  nucleic  acid  or  lecithin." 
The  percentage  of  phosphorus  in  phosphoproteins  is  very  similar  to  that 
in  nucleoproteins  but  they  dift'er  from  this  latter  class  of  proteins  in  that 
they  do  not  yield  any  purine  bases  upon  hydrolytic  cleavage.  Two  of  the 
common  phosphoproteins  are  the  caseinogen  of  milk  and  the  ovovitellin  of 
the  egg-yolk. 

The  hemoglobins  (chromoproteins)  are  compounds  of  the  protein 
molecule  with  haematin  or  some  similar  substance.  The  principal  member 
of  the  group  is  the  haemoglobin  of  the  blood.  Upon  hydrolytic  cleavage 
this  haemoglobin  yields  a  protein  termed  globin  and  a  coloring  matter 
termed  hmnochromogen.  The  latter  substance  contains  iron  and  upon 
coming  in  contact  with  oxygen  is  oxidized  to  form  hcematin.  Hamocyanin, 
another  member  of  the  class  of  haemoglobins,  occurs  in  the  blood  of  certain 
invertebrates,  notably  cephalopods,  gasteropods,  and  Crustacea.  Haemo- 
cyanin  generally  contains  either  copper,  manganese,  or  zinc  in  place  of  the 
iron  of  the  haemoglobin  molecule. 

The  lecithoproteins  include  such  substances  as  lecithans  and  phospha- 
tides which  consist  of  a  protein  molecule  joined  to  lecithin.  They  have 
been  comparatively  little  studied  until  recently,  and  in  much  of  the  older 
research  they  were  undoubtedly  considered  as  lecithins. 

For  experiments  on  conjugated  proteins  see  pages  63,  162,  247,  251, 
271,  and  308. 

DERIVED  PROTEINS. 

These  substances  are  derivatives  which  are  formed  through  hydrolytic 
changes  of  the  original  protein  molecule.  They  may  be  divided  into  two 
groups,  the  primary  protein  derivatives  and  the  secondary  protein  deriva- 
tives. The  term  secondary  derivatives  is  made  use  of  in  this  connection 
since  the  formation  of  the  primary  derivatives  generally  precedes  the 
formation  of  these  secondary  derivatives.  These  derived  proteins  are 
obtained  from  native  simple  proteins  by  hydrolyses  of  various  kinds,  e.  g., 
through  the  action  of  acids,  alkalis,  heat,  or  enzymes.  The  particular 
class  of  derived  protein  desired  regulates  the  method  of  treatment  to  which 
the  native  protein  is  subjected. 

Primary  Protein  Derivatives. 

The  primary  protein  derivatives  are  "apparently  formed  through 
hydrolytic  changes  which  involve  only  slight  alterations  of  the  protein 


PROTEINS. 


molecule."     This  class  includes  proteans,  melaproteins,  and   coagulated 
proteins. 

PROTEANS. 

Proteans  arc  those  insoluble  protein  substances  which  are  produced 
from  proteins  originally  soluble  through  the  incipient  action  of  water, 
enzymes,  or  very  dilute  acids.  It  is  well  known  that  globulins  become 
insoluble  upon  repeated  reprecipitation  and  it  may  possibly  be  found  that 
the  greater  number  of  the  proteans  are  transformed  globulins.  Osborne, 
however,  believes  that  nearly  all  proteins  may  give  rise  to  proteans.  This 
investigator  who  has  so  very  thoroughly  investigated  many  of  the  vege- 
table proteins  claims  that  the  hydrogen  ion  is  the  active  agent  in  the  trans- 
formation. The  protein  produced  from  the  transformation  of  edestin  is 
called  edestan,  that  produced  from  myosin  is  called  myosan,  etc.  The 
name  protean  was  first  given  to  this  class  of  proteins  by  Osborne  in  1900 
in  connection  with  his  studies  of  edestin. 

Experiments  on  Proteans. 

Preparation  and  Study  of  Edestan. — Prepare  edestin  according  to 
the  directions  given  on  page  log.  Bring  the  edestin  into  solution  in 
0.2  per  cent  hydrochloric  acid  and  permit  the  acid  solution  to  stand  for 
about  one-half  hour.^  Neutralize,  with  a  0.5  per  cent  solution  of  sodium 
carbonate,  filter  off  the  precipitate  of  edestan  and  make  the  following 
tests: 

1.  Solubility. — Try  the  solubility  in  the  ordinary  solvents  (see  page 
27).  Note  the  altered  solubility  of  the  edestan  as  compared  with  that  of 
edestin  (see  page  no). 

2.  Millons  Reaction. 

3.  Coagulation  Test. — Place  a  small  amount  of  the  protean  in  a 
test-tube,  add  a  little  water  and  boil.  Now  add  dilute  hydrochloric  acid 
and  note  that  the  protein  no  longer  dissolves.     It  has  been  coagulated. 

4.  Tests  on  Edestan  Solution. — Dissolve  the  remainder  of  the 
edestan  precipitate  in  0.2  per  cent  hydrochloric  acid  and  make  the  follow- 
ing tests: 

(a)  Biuret  Test. 

(b)  Influence  of  Protein  Precipitants. — Try  a  few  protein  precipitants 
such  as  picric  acid  and  mercuric  chloride. 

METAPROTEINS. 

The  metaproteins  are  formed  from  the  native  simple  proteins  through 
an  action  similar  to  that  by  which  proteans  are  formed.     In  the  case  of 

'  The  edestan  solution  preserved  from  experiment  (5),  page  iii,  may  be  used. 


Il6  PHYSIOLOGICAL   CHEMISTRY. 

• 

the  metaproteins,  however,  the  changes  in  the  original  protein  molecule 
are  more  profound.  These  derived  proteins  are  characterized  by  being 
soluble  in  very  weak  acids  and  alkalis,  but  insoluble  in  neutral  fluids. 
The  metaproteins  have  generally  been  termed  albuminates,  but  inasmuch 
as  the  termination  ate  signifies  a  salt  it  has  always  been  somewhat  of  a 
misnomer. 

Two  of  the  principal  metaproteins  are  the  acid  metaprotein  or  so- 
called  acid  albuminate  and  the  alkali  metaprotein  or  so-called  alkali 
albuminate.  They  differ  from  the  native  simple  proteins  principally 
in  being  insoluble  in  sodium  chloride  solution  and  in  not  being  coagula- 
ted except  when  suspended  in  neutral  fluids.  Both  forms  of  metaprotein 
are  precipitated  upon  the  approximate  neutralization  of  their  solutions. 
They  are  precipitated  by  saturating  their  solutions  with  ammonium  sul- 
phate, and  by  sodium  chloride,  also,  provided  they  are  dissolved  in 
an  acid  solution.  Acid  metaprotein  contains  a  higher  percentage  of 
nitrogen  and  sulphur  than  the  alkali  metaprotein  from  the  same  source, 
since  some  of  the  nitrogen  and  sulphur  of  the  original  protein  is 
liberated  in  the  formation  of  the  latter.  Because  of  this  fact,  it  is  impos- 
sible to  transform  an  alkali  metaprotein  into  an  acid  metaprotein,  while  it 
is  possible  to  reverse  the  process  and  transform  the  acid  metaprotein  into 
the  alkali  modification. 

Experiments  on  Metaproteins. 

ACID  METAPROTEm  (ACID  ALBUMINATE). 

Preparation  and  Study. — Take  25  grams  of  hashed  lean  beef  washed 
free  from  the  major  portion  of  blood  and  inorganic  matter,  and  place  it  in 
a  medium-sized  beaker  with  100  c.c.  of  0.2  per  cent  HCl.  Place  it  on  a 
boihng  water-bath  for  one-half  hour,  filter,  cool,  and  divide  the  filtrate 
into  two  parts.  Neutralize  the  first  part  with  dilute  KOH  solution, 
filter  oflf  the  precipitate  of  acid  metaprotein  and  make  the  following  tests: 

(i)  Solubility. — Solubility  in  the  ordinary   solvents    (see  page   27). 

(2)  Milton's  Reaction. 

(3)  Coagulation  Test. — Suspend  a  little  of  the  metaprotein  in  water 
(neutral  solution)  and  heat  to  boiling  for  a  few  moments.  Now  add 
1-2  drops  of  KOH  solution  to  the  water  and  see  if  the  metaprotein  is  still 
soluble  in  dilute  alkali.     What  is  the  result  and  why  ? 

(4)  Test  for  Loosely  Combined  Sulphur  (seepage  108). 

Subject  the  second  part  of  the  original  solution  to  the  following  tests: 
(i)  Coagulation  Test. — Heat  some  of  the  solution  to  boiling  in  a  test- 
tube.     Does  it  coagulate  ? 
(2)  Biuret  Test. 


PROTEINS.  117 

(3)  Influence  of  Protein  Precipitants. — Try  a  few  protein  prccipitants 
such  as  picric  acid  and  mercuric  chloride.  How  do  the  results  obtained 
compare  with  those  from  the  experiments  on  egg  albumin?  (See 
page  102.) 

ALKALI  METAPROTEIN  (ALKALI  ALBUMINATE). 

Preparation  and  Study. — Carefully  separate  the  white  from  the 
yolk  of  a  hcn.'s  egg  and  place  the  former  in  an  evaporating  dish.  Add 
concentrated  potassium  hydroxide  solution,  drop  by  drop,  stirring  con- 
tinuously. The  mass  gradually  thickens  and  finally  assumes  the  con- 
sistency of  jelly.  This  is  solid  alkali  metaprotein  or  "Lieberkiihn's 
jelly."  Do  not  add  an  excess  of  potassium  hydroxide  or  the  jelly  will 
dissolve.  Cut  it  into  small  pieces,  place  a  cloth  or  wire  gauze  over  the 
dish,  and  by  means  of  running  water  wash  the  pieces  free  from  adherent 
alkali.  Now  add  a  small  amount  of  water,  which  forms  a  weak  alkaline 
solution  with  the  alkali  within  the  pieces,  and  dissolve  the  jelly  by  gentle 
heat.  Cool  the  solution  and  divide  it  into  two  parts.  Proceed  as  follows 
with  the  first  part:  Neutralize  with  dilute  hydrochloric  acid,  noting  the 
odor  of  the  liberated  hydrogen  sulphide  as  the  alkali  metaprotein  precipi- 
tates. Filter  off  the  precipitate  and  test  as  for  acid  metaprotein,  page 
116,  noting  particularly  the  sulphur  test.  How  does  this  test  compare  with 
that  given  by  the  acid  metaprotein?  Make  tests  on  the  second  part  of 
the  solution  the  same  as  for  acid  metaprotein,  page  116. 

Coagulated  Proteins. 

These  derived  proteins  are  produced  from  unaltered  protein  mate- 
rials by  heat,  by  long  standing  under  alcohol,  or  by  the  continuous  move- 
ment of  their  solutions  such  as  that  produced  by  rapid  stirring  or  shaking. 
In  particular  instances,  such  as  the  formation  of  fibrin  from  fibrinogen 
(see  page  195),  the  coagulation  may  be  produced  by  enzyme  action. 
Ordinary  soluble  proteins  after  having  been  transformed  into  the  coagu- 
lated modification  are  no  longer  soluble  in  the  ordinary  solvents.  Upon 
being  heated  in  the  presence  of  strong  acids  or  alkalis,  coagulated  proteins 
are  converted  into  metaproteins. 

Many  proteins  coagulate  at  an  approximately  fixed  temperature  under 
definite  conditions  (see  pp.  106  and  254).  This  characteristic  may  be 
applied  to  separate  different  coagulable  proteins  from  the  same  solution 
by  fractional  coagulation.  The  coagulation  temperature  frequently  may 
serve  in  a  measure  to  identify  proteins  in  a  manner  similar  to  the  melting- 
point  or  boiling-point  of  many  other  organic  substances.  The  separation 
of  proteins  by  fractional  coagulation  is  thus  analogous  to  the  separation 
of  volatile  substances  by  means  oi  fractional  distillation.     This  method  of 


Il8  PHYSIOLOGICAL    CHEMISTRY. 

separating  proteins  is  not  a  satisfactory  one,  however,  inasmuch  as  proteins 
in  solution  have  different  effects  upon  one  another  and  also  because  of  the 
fact  that  the  nature  of  the  solvent  causes  a  variation  in  the  temperature  at 
which  a  given  protein  coagulates.  The  nature  of  the  process  involved  in 
the  coagulation  of  proteins  by  heat  is  not  well  understood,  but  it  is  probable 
that  in  addition  to  the  altered  arrangement  of  the  component  atoms  in  the 
molecule,  there  is  a  mild  hydrolysis  which  is  accompanied  by  the  libera- 
tion of  minute  amounts  of  hydrogen,  nitrogen,  and  sulphur.  The  pres- 
ence of  a  neutral  salt  or  a  trace  of  a  mineral  acid  may  facilitate  the  coagu- 
lation of  a  protein  solution  (see  page  io6),  whereas  any  appreciable 
amount  of  acid  or  alkali  will  retard  or  entirely  prevent  such  coagulation. 

It  has  recently  been  shown  that  the  coagulation  of  proteins  by  heat 
proceeds  in  two  stages,^  first,  a  reaction  between  the  protein  and  the  hot 
water  (denaturation)  and  second,  an  agglutination  or  separation  of  the 
altered  protein  in  particulate  form.  The  concentration  of  acid,  or  hydro- 
gen ion,  in  the  solution  influences  the  coagulation  of  proteins,  such  that 
the  original  protein  is  acted  upon  less  readily  by  hot  water  alone  than  in  the 
presence  of  acid.  The  formation  of  the  coagulum  is  accompanied  by  the 
disappearance  of  the  free  acid  from  the  solution,  indicating  the  formation 
of  a  protein  salt.  A  disturbance  of  the  equilibrium  between  the  hydro- 
lyzed  and  unhydrolyzed  portions  of  the  protein  salt,  due  to  the  greater 
rapidity  with  which  the  unhydrolyzed  portion  is  precipitated,  results  in 
the  gradual  removal  of  both  protein  and  acid  from  the  solution.  This 
has  been  offered  as  an  explanation  of  the  decreasing  acidity. 

According  to  Chick  and  Martin,  the  addition  of  neutral  salts  to  the 
acid  solution  of  the  salt-free  protein  to  be  coagulated  results  in  a  decreased 
rate  of  coagulation.  This  is  due  in  part  to  the  decrease  in  the  concen- 
tration of  the  free  acid,  which  results  from  the  disturbance  of  the  equilib- 
rium between  the  protein  and  acid  and  also  in  part  to  the  direct  influence 
which  the  salts  exert  upon  the  protein.  The  presence  of  neutral  salts 
may  under  certain  circumstances  facilitate  the  coagulation  of  proteins  by 
heat. 

The  temperature  at  which  egg  white  is  coagulated  causes  a  difference 
in  the  appearance  of  the  coagulum.^  Coagulated  egg  white  which  has 
been  immersed  in  water  at  a  low  temperature  and  then  gradually  heated 
to  the  coagulating  temperature  is  more  translucent  and  has  a  bluish  color, 
whereas,  egg  white  which  has  been  immersed  in  water  heated  to  a  tem- 
perature above  the  coagulating  temperature  is  creamy-white  in  color. 
The  varying  digestibitily,  as  the  result  of  the  different  methods  of  heating, 
has  been  discussed  in  the  chapter  on  Enzymes. 

'  Chick  and  Martin:   Journal  of  Physiology,  43,  i,  1911. 
'■'P'rank:    Journal  of  Biological  Chemistry,  9,  463,  191 1 


proteins.  119 

Experiments  on  Coagulated  Protein. 

Ordinary  coagulated  egg-white  may  be  used  in  the  following  tests: 

1.  Solubility. — Try  the  solubility  of  small  pieces  of  the  coagulated 
protein  in  each  of  the  ordinary  sclvents  (see  page  27). 

2.  Millon's  Reaction. 

3.  Xanthoproteic  Reaction. — Partly  dissolve  a  medium-sized  piece 
of  the  protein  in  concentrated  nitric  acid.  Cool  the  solution  and  add  an 
excess  of  ammonium  hydroxide.  Both  the  protein  solution  and  the 
undissolved  protein  will  be  colored  orange. 

4.  Biuret  Test. — Partly  dissolve  a  medium-sized  piece  of  the  protein 
in  concentrated  potassium  hydroxide  solution.  If  the  proper  dilution  of 
copper  sulphate  solution  is  now  added  the  white  coagulated  protein,  as 
well  as  the  protein  solution,  will  assume  the  characteristic  purplish-violet 
color. 

5.  Hopkin's-Cole  Reaction.— Conduct  this  test  according  to  the 
moditication  given  on  page  98. 

Secondary  Protein   Derivatives. 

These  derivatives  result  from  a  more  profound  cleavage  of  the  protein 
molecule  than  that  which  occurs  in  the  formation  of  the  primary  deriva- 
tives.    The  class  includes  proteoses,  peptanes,  and  peptides. 

PROTEOSES  AND  PEPTONES. 

Proteoses  are  intermediate  products  in  the  digestion  of  proteins  by 
proteolytic  enzymes,  as  well  as  in  the  decomposition  of  proteins  by  hydrol- 
ysis and  the  putrefaction  of  proteins  through  the  action  of  bacteria. 
Proteoses  are  called  albiimoses  by  some  writers,  but  it  seems  more  logical 
to  reserve  the  term  ablumose  for  the  proteose  of  albumin. 

Peptones  are  formed  after  the  proteoses  and  it  has  been  customary  to 
consider  them  as  the  last  product  of  the  processes  before  mentioned  which 
still  possess  true  protein  characteristics.  In  other  words  it  has  been 
considered  that  the  protein  nature  of  the  end-products  of  the  cleavage  of 
the  protein  molecule  ceased  with  the  peptones,  and  that  the  simpler 
bodies  formed  from  peptones  were  substances  of  a  different  nature  (see 
page  70).  However,  as  the  end-products  have  been  more  carefully 
studied,  it  has  been  found  to  be  no  easy  matter  to  designate  the  exact 
character  of  a  peptone  or  to  indicate  the  exact  point  at  which  the  peptone 
characteristic  ends  and  the  peptide  characteristic  begins.  The  situation 
regarding  the  proteoses,  peptones  and  peptides,  is  at  present  a  most 
unsatisfactory  one  because  of  the  unsettled  state  of  our  knowledge  regard- 
ing them.     The  exact  differences  between  certain  members  of  the  peptone 


I20  PHYSIOLOGICAL   CHEMISTRY. 

and  peptide  groups  remain  to  be  more  accurately  established.  It  has  been 
quite  well  established  that  the  peptones  are  peptides  or  mixtures  of  peptides 
but  the  term  peptide  is  used  at  present  to  designate  only  those  possessing  a 
definite  structure. 

There  are  several  proteoses  (protoproteose,  heteroproteose  and  deutero- 
proteose),  and  at  least  two  peptones  (amphopeptone  and  antipeptone), 
which  result  from  proteolysis.  The  differentiation  of  the  various  proteoses 
and  peptones  at  present  in  use  is  rather  unsatisfactory.  These  compounds 
are  classified  according  to  their  varying  solubihties,  especially  in  ammo- 
nium sulphate  solutions  of  different  strengths.  The  exact  differences  in 
composition  between  the  various  members  of  the  group  remain  to  be  more 
accurately  established.  Because  of  the  difficulty  attending  the  separation 
of  these  bodies,  pure  proteose  and  peptone  are  not  easy  to  procure.  The 
so-called  peptones  sold  commercially  contain  a  large  amount  of  proteose. 
As  a  class  the  proteoses  and  peptones  are  very  soluble,  diffusible  bodies 
which  are  non-coagulable  by  heat.  Peptones  differ  from  proteoses  in  being 
more  diffusible,  non-precipitable  by  (NH4)2SO^,  and  by  their  failure  to  give 
any  reaction  with  potassium  ferrocyanide  and  acetic  acid,  potassio-mer curie 
iodide  and  HCl,  picric  acid,  and  trichloracetic  acid.  The  so-called  primary 
proteoses  are  precipitated  by  HNO3  and  are  the  only  members  of  the  pro- 
teose-peptone  group  which  are  so  precipitated. 

Some  of  the  more  general  characteristics  of  the  proteose-peptone 
group  may  be  noted  by  making  the  following  simple  tests  on  a  proteose- 
peptone  powder: 

(i)  Solubility. — Solubihty  in   the   ordinary   solvents    (see  page   27). 

(2)  Millon^s  Reaction. 

Dissolve  a  little  of  the  powder  in  water  and  test  the  solution  as 
follows: 

(i)  Precipitation  by  Picric  Acid. — To  5  c.c.  of  proteose-peptone 
solution  in  a  test-tube  add  picric  acid  until  a  permanent  precipitate  forms. 
The  precipitate  disappears  on  heating  and  returns  on  cooling. 

(2)  Precipitation  by  a  Mineral  Acid.- — Try  the  precipitation  by  nitric 
acid. 

(3)  Coagulation  Test. — Heat  a  little  proteose-peptone  solution  to  boil- 
ing.    Does  it  coagulate  like  the  other  simple  proteins  studied? 

SEPARATION  OF  PROTEOSES  AND  PEPTONES." 

Place  50  c.c.  of  proteose-peptone  solution  in  an  evaporating  dish  or 
casserole,  and  half-saturate  it  with  ammonium  sulphate  solution,  which 

'  The  separation  of  proteoses  and  peptones  by  means  of  fractional  precipitation  with 
ammonium  sulphate  does  not  possess  the  significance  it  was  once  supiposed  to  possess  inas- 
much as  the  boundary  between  these  substances  and  peptides  is  not  well  defined  (see  p.  119). 


PROTEINS.  121 

may  be  accomplished  by  adding  an  equal  volume  of  saturated  ammonium 
sulphate  solution.  At  this  point  note  the  appearance  of  a  precipitate  of 
the  primary  proteoses  (protoproteose  and  hctero-proteose).  Now  heat  the 
half-saturated  solution  and  its  suspended  precipitate  to  boiling  and 
saturate  the  solution  with  solid  ammonium  sulphate.  At  full  saturation  the 
secandary  proteoses  (deuteroproteoses)  are  precipitated.  The  peptones 
remain  in  solution. 

Proceed  as  fallows  with  the  precipitate  of  proteoses:  Collect  the  sticky 
precipitate  on  a  rubber-tipped  stirring  rod  or  remove  it  by  means  of  a 
watch  glass  to  a  small  evaporating  dish  and  dissolve  it  in  a  little  water. 
To  remove  the  ammonium  sulphate,  which  adhered  to  the  precipitate 
and  is  now  in  solution,  add  barium  carbonate,  boil,  and  filter  off  the 
precipitate  of  barium  sulphate.  Concentrate  the  proteose  solution  to  a 
small  volume^  and  make  the  following  tests: 

(i)  Biuret  Test. 

(2)  Precipitatian  by  Nitric  Acid. — A\Tiat  would  a  precipitate  at  this 
point  indicate  ? 

(3)  Precipitatian  by  Trichloracetic  Acid. — This  precipitate  dissolves 
on  heating  and  returns  on  cooling. 

(4)  Precipitatian  by  Picric  Acid. — This  precipitate  also  disappears  on 
heating  and  returns  on  cooling. 

(5)  Precipitation  by  Potassio-mer curie  Iodide  and  Hydrochloric  Acid. 

(6)  Coagulatian  Test. — Boil  a  little  in  a  test-tube.     Does  it  coagulate  ? 

(7)  Acetic  Acid  and  Potassium  Ferrocyanide  Test. 

The  solution  containing  the  peptones  should  be  cooled  and  filtered, 
and  the  ammonium  sulphate  in  solution  removed  by  boiling  with  barium 
carbonate  as  described  above.  After  filtering  off  the  barium  sulphate 
precipitate,  concentrate  the  peptone  filtrate  to  a  small  volume  and  repeat 
the  test  as  given  under  the  proteose  solution,  above.  In  the  biuret  test 
the  solution  should  be  made  very  strongly  alkaline  with  solid  potassium 
hydroxide. 

PEPTIDES. 

The  peptides  are  "definitely  characterized  combinations  of  two  or 
more  amino  acids,  the  carboxyl  (COOH)  group  of  one  being  united 
with  the  amino  (NHj)  group  of  the  other  with  the  elimination  of  a  mole- 
cule of  water."  These  peptides  are  more  fully  discussed  on  pages  71 
and  119, 

*  If  the  proteoses  are  desired  in  powder  form,  this  concentrated  proteose  solution  may 
now  be  precipitated  by  alcohol,  and  this  precipitate,  after  being  washed  with  absolute  alcohol 
and  with  ether,  may  be  dried  and  powdered. 


122  PHYSIOLOGICAL    CHEMISTRY. 

REVIEW  OF  PROTEINS. 

In  order  to  facilitate  the  student's  review  of  the  proteins,  the  prepara 
tion  of  a  chart  similar  to  the  model  given  is  recommended.     The  signs  + , 
and  —  may  be  conveniently  used  to  indicate  positive  and  negative  reactions. 

MODEL  CHART  FOR  REVIEW  PURPOSES. 


Protein. 

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"Unknov^n"  Mixtures  and  Solutions  of  Proteins. 

At  this  point  the  student's  knowledge  of  the  characteristics  of  the 
various  proteins  studied  will  be  tested  by  requiring  him  to  examine  several 
'•'unknown"  protein  mixtures  or  solutions  and  make  full  report  upon  the 
same.     The  scheme  given  on  page  123  may  be  used  in  this  examination. 


PROTEINS. 


123 


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CHAPTER  VI. 
GASTRIC  DIGESTION. 

Gastric  digestion  takes  place  in  the  stomach  and  is  promoted  by 
the  gastric  juice,  which  is  secreted  by  the  glands  of  the  stomach  mucosa. 
These  glands  are  of  two  kinds,  fundus  glands  and  pyloric  glands  which 
are  situated,  as  their  names  imply,  in  the  regions  of  the  fundus  and 
pylorus.  The  principal  foods  acted  upon  in  gastric  digestion  are  the 
proteins  which  are  so  changed  by  its  processes  as  to  become  better  pre- 
pared for  further  digestion  in  the  intestine  and  for  their  final  absorption. 

From  reliable  experiments  made  upon  lower  animals  it  is  evident 
that  the  gastric  juice  is  secreted  as  the  result  of  stimuli  of  two  forms, 
i.  e.,  psychical  stimuli  and  chemical  stimuli.  The  psychical  form  of 
stimuli  may  be  produced  by  the  sight,  thought,  or  taste  of  food,  and  the 
chemical  stimuli  may  be  produced  by  certain  substances,  such  as  water, 
milk,  the  extractives  of  meat,  etc.,  when  coming  in  contact  with  the 
stomach  mucosa.  The  stimulatory  power  of  water  has  been  very  strik- 
ingly demonstrated/  Experiments  have  been  made  which  indicate 
clearly  that  the  outpouring  of  gastric  juice  increases  in  direct  proportion 
to  the  volume  of  water  which  comes  into  contact  with  the  gastric  mucosa.^ 
The  claim  that  the  drinking  of  water  with  meals  is  harmful  because  such 
a  procedure  causes  a  dilution  of  the  gastric  juice,  has  no  basis  in  fact. 
The  drinking  of  water  with  meals  by  normal  individuals  has  been  found 
to  be  accompanied  by  a  more  economical  utilization  of  the  ingested  pro- 
teins, fats  and  carbohydrates.  Various  other  desirable  and  no  undesirable 
features  have  been  demonstrated  as  accompanying  or  following  such  a 
dietary  procedure.^  No  experimental  evidence  has  been  submitted  which 
can  justly  be  interpreted  as  showing  any  harmful  influence  to  accompany 
or  follow  the  drinking,  by  normal  persons,  of  large  quantities  of  water  at 
meal  time. 

*  Foster  and  Lambert:  Journ  Exper.  Med.,  lo,  820,  1908. 

2  Wills  and  Hawk:  Jour.  Biol.  Chem.,  9,  xxx,  191 1.     (Proceedings). 

'Hawk:  University  0/ Pennsylvania  Medical  Bulletin,  18,  i,  1905. 

Fowler  and  Hawk:  Jour.  Exper.  Med.,  12,  388,  1910. 

Hattrem  and  Hawk:  Arch.  Int.  Med.,  7,  610,  1911. 

Mattill  and  Hawk:  Jour.  Am.  Chem.  Soc,  33,  pp.  1978,  1999,  and  2019,  1911. 

Hawk:  Arch.  Int.  Med.,  8,  382,  1911. 

Hawk:  Proceedings  Soc.  Exp.  Biol,  and  Med.,  8,  36,  1910. 

Fairhall  and  Hawk:  Jour.  Am.  Chem.  Soc,  34,  546,  1912. 

Howe  and  Hawk:  Jour.  Biol.  Chem.,  11,  129,  1912. 

124 


GASTRIC   DIGESTION.  1 25 

The  volume  of  gastric  juice  secreted  during  any  given  period  of 
digestion,  \aries  with  the  quantity  and  kind  of  the  food.  These  con- 
clusions were  deduced  principally  from  a  series  of  so-called  delusive 
feeding  experiments.  A  dog  was  prepared  with  two  oesophageal  openings 
and  a  gastric  fistula.  When  thus  prepared  and  fed  foods  of  various  kinds 
such  as  meat  and  bread,  the  material  instead  of  passing  to  the  stomach, 
would  invariably  find  its  way  out  of  the  animal's  body  at  the  upper 
oesophageal  opening.  Through  the  medium  of  the  gastric  fistula  the 
course  of  the  secretion  of  gastric  juice  could  be  carefully  followed.  It 
was  found  that  when  the  dog  ate  meat,  for  example,  there  was  a  large 
secretion  of  gastric  juice  notwithstanding  no  portion  of  the  food  eaten 
had  reached  the  stomach.  Further  experiments  made  through  the 
medium  of  a  cul-de-sac  formed  from  the  stomach  wall  have  given  us 
many  valuable  conclusions,  among  others  those  regarding  the  influence 
of  the  chemical  stimuli.  The  method  followed  was  to  feed  the  animal 
certain  substances  and  note  the  secretion  of  gastric  juice  in  the  miniature 
stomach  while  the  real  process  of  digestion  was  taking  place  in  the 
stomach  proper. 

Normal  gastric  juice  is  a  thin,  light  colored  fluid  which  is  acid  in 
reaction  and  has  a  specific  graA^ity  varying  between  i.ooi  and  i.oio. 
It  contains  only  2-3  per  cent  of  solid  matter  which  is  made  up  prin- 
cipally of  hydrochloric  acid,  sodium  chloride,  potassium  chloride,  earthy 
phosphates,  mucin  and  the  enzymes  pepsin,  gastric  rennin,  and  gastric 
lipase;  the  hydrochloric  acid  and  the  enzymes  are  of  the  greatest  im- 
portance. The  acidity  of  the  gastric  juice  is  due  to  free  hydrochloric. 
It  was  formerly  believed  that  this  acid  was  secreted  by  the  parietal  cells 
of  the  fundus  as  well  as  by  the  chief  cells  of  both  the  fundus  and  pyloric 
glands.  It  has  recently  been  claimed^  however,  that  the  parietal  cell  is 
the  seat  of  the  formation  of  the  hydrochloric  acid.  This  conclusion  is  based 
upon  the  formation  of  Prussian  blue  after  the  subcutaneous  injection  of 
potassium  ferrocyanide  and  ammonium  ferric  citrate  (rabbits  and 
guinea-pigs)  and  the  subsequent  (3  to  30  hours)  microscopical  examina- 
tion of  the  gastric  mucosa.  The  acid  was  shown  to  be  present  in  the 
lumina  of  the  gland  tubules  and  in  the  canaliculi  of  the  parietal  cells; 
traces  were  also  apparently  present  in  the  cytoplasm.  Still  more  recently 
Bensley  and  Harvey'  have  shown  by  means  of  dyes  which  act  as  vital 
stains  and  as  indicators  very  sensitive  to  alkali  that  the  secretion  in  the 
parietal  cells  is  slightly  alkaline  whereas  that  in  the  lumen  of  the  gland 
proper  is  very  nearly  neutral.  Therefore,  the  acid  is  formed  entirely  above 
the  level  of  the  gland  proper  i.  e.  in  the  foveolae  and  on  the  surface.     It  is 

'  Fitzgerald:  Proceedings  Royal  Society  (B),  83,  56,  igio. 

-  Bensley  and  Harvey:  Unpublished  data  furnished  by  Dr.  Bensley. 


126  PHYSIOLOGICAL    CHEMISTRY. 

apparent  from  the  work  of  Fitzgerald,  and  Bensley  and  Harvey  that 
the  question  as  to  the  seat  of  formation  of  the  hydrochloric  acid  must  be 
considered  as  undecided. 

Hydrochloric  acid  is  generally  present  in  the  gastric  juice  of  man  to 
the  extent  of  o .  2-0 . 3  per  cent.  When  the  amount  of  hydrochloric  acid 
varies  to  any  considerable  degree  from  these  values  a  condition  of 
hypoacidity  or  hyperacidity  is  established.  Hydrochloric  acid  has  the 
power  of  combining  with  protein  substances  taken  in  the  food-,  thus 
forming  so-called  combined  hydrochloric  acid.  This  combined  acid  is  a 
less  potent  germicide  than/ree  hydrochloric  acid  and  has  less  power  to 
destroy  the  amylolytic  enzyme  salivary  amylase  (ptyalin)  of  the  saliva. 
This  last  fact  explains  to  a  degree  the  possibility  of  the  continuance  of 
salivary  digestion  in  the  stomach. 

The  term  combined  hydrochloric  acid  is  really  a  misnomer.  When 
free  hydrochloric  acid  is  treated  with  a  protein  the  latter  functions  as  a 
base  metal  and  a  salt  is  formed.  Therefore,  instead  of  having,  "com- 
bined hydrochloric  acid"  we  have  a  protein  salt  of  hydrochloric  acid. 
This  salt  ionizes  differently  from  the  free  acid.  This  fact  explains  the 
variation  in  the  gemicidal  properties  of  the  two  solutions  as  well  as  their 
different  action  toward  enzymes,  such,  for  example,  as  salivary  amylase 
(see  page  66). 

The  hydrochloric  acid  of  the  gastric  juice  forms  a  medium  in  which 
the  pepsin  can  most  satisfactorily  digest  the  protein  food,  and  at  the 
same  time  it  acts  as  an  antiseptic  or  germicide  which  prevents  putre- 
factive processes  in  the  stomach.  It  also  possesses  the  power  of  inverting 
cane  sugar,  this  property  being  due  to  the  hydrogen  ion.  When  the 
hydrochloric  acid  of  the  gastric  juice  is  diminished  in  quantity  (hypoacid- 
ity) or  absent,  as  it  may  be  in  many  cases  of  functional  or  organic  dis- 
ease, there  is  no  check  to  the  growth  of  micro-organisms  in  the  stomach. 
There  are,  however,  certain  of  the  more  resistant  spores  which  even  the 
normal  acidity  of  the  gastric  juice  will  not  destroy.  A  condition  of 
hypoacidity  may  also  give  rise  to  fermentation  with  the  formation  of 
comparatively  large  amounts  of  such  substances  as  lactic  acid  and  butyric 
acid. 

The  f|uestion  of  the  origin  of  the  hydrochloric  acid  of  the  gastric 
juice  is  a  problem  to  whose  solution  many  investigators  have  given 
much  attention.  Many  theories  have  been  proposed,  among  them 
being  Bunge's  mass  action  theory,  Koppe's  electrolytic  dissociation  theory. 
and  the  more  recent  theory  based  upon  the  interaction  of  sodium  chloride 
and  lactic  acid.  We  cannot  go  into  a  discussion  of  these  various  theories. 
Each  of  them  has  met  with  objection  and  we  have,  as  yet,  no  generally 
accepted  theory  as  to  the  origin  of  the  hydrochloric  acid  of  the  gastric 


GASTRIC   DIGESTION.  1 27 

juice.  That  this  hydrochloric  acid  originates  from  the  chlorides  of  the 
blood  is  apparently  a  well  established  fact,  but  farther  than  this  no  positive 
statement  can  be  made. 

The  most  important  of  the  enzymes  of  the  f^astric  juice  is  the  pro- 
teolytic enzyme  pepsin.  The  pepsin  does  not  originate  as  such  in  the 
gastric  cells  but  is  formed  from  its  precursor,  the  zymogen  or  mother- 
substance  pepsinogen,  which  is  produced  by  the  parietal  cells  of  the 
fundus  as  well  as  by  the  chief  cells  of  the  fundus  and  pyloric  glands. 
Pepsinogen  may  be  differentiated  from  pepsin  from  the  fact  that  it  is 
more  resistant  to  alkali.^  Upon  coming  in  contact  with  the  hydrochloric 
acid  of  the  secretion  this  pepsinogen  is  immediately  transformed  into 
pepsin.  Pepsin  is  not  active  in  alkaline  or  neutral  solutions  but  requires 
at  least  a  faint  acidity  before  it  can  exert  its  power  to  dissolve  and  digest 
proteins.  The  percentage  of  hydrochloric  acid  facilitating  the  most 
rapid  peptic  action  varies  with  the  character  of  the  protein  acted  upon, 
e.  g.,  0.08  per  cent  to  o.i  per  cent  for  the  digestion  of  fibrin  and  0.25 
per  cent  for  the  digestion  of  coagulated  egg-white.  While  hydrochloric 
acid  is  the  acid  usually  employed  to  promote  artificial  peptic  proteolysis, 
other  acids,  organic  and  inorganic,  will  serve  the  same  purpose.  Acidity 
of  the  liquid  is  necessary  to  promote  the  activity  of  the  pepsin,  but  the 
acidity  need  not  necessarily  be  confined  to  hydrochloric  acid. 

In  common  with  many  other  enzymes  pepsin  acts  best  at  about 
38^-40°  C.  and  its  digestive  power  decreases  as  the  temperature  is  lowered, 
the  enzyme  being  only  slightly  active  at  o°  C.  Its  power  is  only  tempo- 
rarily inhibited  by  the  appHcation  of  such  low  temperatures,  however, 
and  the  enzyme  regains  its  full  proteolytic  power  upon  raising  the  tem- 
perature to  40°  C.  As  the  temperature  of  a  digestive  mixture  is  raised 
above  40°  C.  the  pepsin  gradually  loses  its  activity  until  at  about  80°- 
100°  C.  its  proteolytic  power  is  permanently  destroyed. 

Our  ideas  regarding  the  nature  of  the  products  formed  in  the  course 
of  peptic  proteolysis  have  undergone  considerable  revision  in  recent 
years.  The  former  view  that  these  products  included  only  acid  albu- 
minate (acid  metaprotein),  proteoses  and  peptones  is  no  longer  tenable. 
From  the  investigations  of  numerous  observers  we  have  learned  that 
artificial  gastric  digestion  if  permitted  to  proceed  for  a  sufjiciently  long 
period  will  yield,  in  addition  to  proteoses  and  peptones,  a  long  list  of 
protein  cleavage  products  which  are  crystalline  in  character,  including 
leucine,  tyrosine,  alanine,  phenylalanine,  aspartic  acid,  glutamic  acid, 
proline,  leucininiide,  valine,  and  lysine.  A  similar  group  of  substances 
may  result  from  the  action  of  the  enzyme  trypsin  (see  p.  149).  The 
relative    amounts    of    proteoses,  peptones,    and    crystalline    substances 

'Langley:  Jour,  of  Physiol.,  3.  p.  246. 


128  PHYSIOLOGICAL    CHEMISTRY. 

formed  depends  to  a  great  extent  upon  the  character  of  the  protein  under- 
going digestion,  e.  g.,  a,  greater  proportion  of  proteoses  results  from  the 
digestion  of  fibrin  than  from  the  digestion  of  coagulated  egg-white.  We 
must  not  be  led  into  the  error  of  thinking  that  the  large  number  of  protein 
cleavage  products  just  mentioned  are  formed  in  the  course  of  normal 
gastric  digestion  within  the  animal  organism.  They  are  formed  only 
after  comparatively  long-continued  hydrolysis.  In  pancreatic  digestion, 
however,  there  are  formed  even  under  normal  conditions,  the  .large 
number  of  cleavage  products  to  which  reference  has  been  made.  Peptic 
proteolysis,  therefore,  within  the  animal  organism  differs  from  tryptic 
proteolysis  (see  page  149)  in  that  the  former  yields  larger  amounts  of 
proteoses,  smaller  amounts  of  peptones  and  no  considerable  quantity 
of  crystalline  bodies  as  end-products  in  the  brief  period  during  which 
proteins  are  ordinarily  subjected  to  gastric  digestion.  Prolonged  hydrol- 
ysis with  gastric  juice  does,  however,  yield  considerable  quantities  of 
the  non-protein  end-products.  In  cases  of  cancer  of  the  stomach  a 
peptide-splitting  enzyme  (erepsin)  is  present  in  the  stomach  contents. 
This  enzyme  is  believed  to  be  elaborated  by  the  cancer  tissue  and  its 
identification  is  of  importance  in  connection  with  the  diagnosis  of  gastric 
cancer.  The  glycyl-tryptophane  test^  is  used  for  this  purpose 
(see  p.  15). 

Abderhalden  and  Meyer^  have  very  recently  shown  active  pepsin  to 
be  present  in  the  contents  of  all  parts  of  the  small  intestine.  It  is  suggested 
that  pepsin  may  be  adsorbed  in  the  stomach  by  such  protein  substances 
as  pass  into  the  intestine  in  solid  form  and  that  the  pepsin  thus  protected 
may  bring  about  gastric  digestion  whenever  the  reaction  of  the  surrounding 
intestinal  contents  is  favorable.  This  fact  may  be  of  importance  in 
connection  with  the  profound  proteolysis  taking  place  in  the  intestine. 
Heretofore,  this  process  was  believed  to  be  furthered  alone  by  trypsin 
and  erepsin.  The  passage  of  adsorbed  pepsin  into  the  intestine  may  be 
an  efficient  aid  to  the  proper  digestion  of  solid  proteins  which  are  ingested 
without  sufficient  mastication  ("bolted")^  and  which  consequently,  at 
times,  pass  into  the  intestine  in  rather  large  pieces  (see  chapter  on  Feces). 

Gastric  rennin,  the  second  enzyme  of  the  gastric  juice,  is  what  is 
known  as  a  milk  curdling  or  protein  coagulating  enzyme.  Rennin  acts 
upon  the  caseinogen  of  the  milk,  splitting  it  into  a  proteose-like  body 
and  soluble  casein.  This  soluble  body,  in  the  presence  of  calcium 
salts,  combines  with  calcium,  forming  calcium  casein  or  true  casein 
which  is  insoluble  and  precipitates.     There  is  some   uncertainty  re- 

'  Neubauer  and  Fischer:  Deut.  Arch./,  klin.  Med.,  97,  499,  1909. 
*  Abderhalden  and  Meyer:  Zeit.fur  physiol.  Chem.,  74,  67,  191 1. 

'  Foster  and  Hawk:  Proceedings  of  the  Eighth  International  Congress  of  Applied  Chemistry, 
New  York,  September,  1912. 


GASTRIC    DIGESTION.  1 29 

garding  the  reaction  to  litmus  in  which  gastric  rcnnin  shows  the  greatest 
activity.  It  is,  however,  said  to  be  active  in  neutral,  alkaline,  or  acid 
solution.  However,  it  probal^ly  possesses  its  greatest  activity  in  the 
presence  of  a  slight  acid  reaction,  as  would  naturally  be  expected.  It 
is  especially  abundant  in  the  gastric  mucosa  of  the  calf,  and  is  used 
to  curdle  the  milk  used  in  cheese-making.  Gastric  rennin  is  always 
present  normally  in  the  gastric  juice  but  in  certain  pathological  con- 
ditions such  as  atrophy  of  the  mucosa,  chronic  catarrh  of  the  stomach, 
or  in  carcinoma  it  may  be  absent. 

The  theory  that  the  proteolytic  activity  and  the  milk  curdling  property 
of  the  gastric  juice  reside  in  a  single  substance  is  causing  much  con- 
troversy at  the  present  time.  The  theory  was  originally  advanced  by  the 
Pawlow  school.^  According  to  Nencki  and  Sieber^  the  milk  curdling 
and  protein  hydrolyzing  activities  reside  in  defmite  and  distinct  side  chains 
of  a  single  mammoth  molecule.  The  view  which  has  rather  the  strongest 
support,  however,  is  to  the  effect  that  there  are  two  entirely  distinct 
enzymes.  Important  evidence  has  been  advanced  in  favor  of  this  view 
by  Hammarsten,^  Taylor,^  and  Hemmeter.^  Very  recently  Burge"  has 
reported  experiments  upon  the  influence  of  a  direct  electric  current  upon 
solutions  possessing  typical  rennin  and  peptic  activities.  By  this  means 
he  was  able  to  prepare  a  solution  possessing  strong  rennin  activity  but 
entirely  void  of  peptic  activity.  This  furnishes  strong  evidence  against 
the  identity  of  the  two  enzymes  but  does  not  necessarily  deny  the  accuracy 
of  the  side-chain  theory. 

Gastric  lipase,  the  third  enzyme  of  the  gastric  juice,  is  a  fat-splitting 
enzyme.  It  possesses  but  slight  activity  when  the  gastric  juice  is  of 
normal  acidity,  but  evinces  its  action  principally  at  such  times  as  a 
gastric  juice  of  low  acidity  is  secreted  either  from  physiological  or  patho- 
logical cause.  The  digestion  of  fat  in  the  stomach  is,  however,  at  most, 
of  but  slight  importance  as  compared  with  the  digestion  of  fat  in  the 
intestine  through  the  action  of  the  lipase  of  the  pancreatic  juice  (see 
page  151). 

PREPARATION  OF  AN  ARTIFICIAL  GASTRIC  JUICE. 

Dissect  the  mucous  membrane  of  a  pig's  stomach  from  the  mus- 
cular portion  and  discard  the  latter.  Divide  the  mucous  membrane 
into  two  parts  (4/5  and  1/5).     Cut  up  the  larger  portion,  place  it  in 

'  Tawlow  and  Parastschuk:  Zeitschrift  fiir  Physiologische  Chemie,  42,  415,  1904. 

-  Xencki  and  Sieber:  Zeitschrift  fiir  Physiologische  Chemie,  23,  2gi,  1901. 

'  Ilammarsten:  Zeitschrift  fiir  Physiologische  C  hemic,  56,  18,  1908. 

*  Taylor:  Journal  of  Biological  C  hemistry,  5,  399,  1909. 

^  Hemmeter:  Berliner  klinische  Wochenschrift,  Ewald  Festnummer,  44,  1905. 

^  Burge:  American  Journal  of  Physiology,  29,  1912. 


130  PHYSIOLOGICAL   CHEMISTRY. 

a  large-sized  beaker  with  0.4  per  cent  hydrochloric  acid  and  keep  at 
38°-4o°  C.  for  at  least  24  hours.  Filter  off  the  residue,  consisting  prin- 
cipally of  nuclein  and  anti-albumid,  and  use  the  filtrate  as  an  artificial 
gastric  juice.  This  filtrate  contains  pepsin,  rennin,  and  the  products 
of  the  digestion  of  the  stomach  tissuue,  i.  e.,  acid  metaprotein  (acid 
albuminate),  proteoses,  peptones,  etc. 

Preparation  of  a  Glycerol  Extract  of  Pig's  Stomach. 

Take  the  one-fifth  portion  of  the  mucous  membrane  of  the  pig's 
stomach  not  used  in  the  preparation  of  the  artificial  gastric  juice,  cut 
it  up  very  finely,  place  it  in  a  small-sized  beaker  and  cover  the  mem- 
brane with  glycerol.  Stir  frequently  and  allow  to  stand  at  room  tem- 
perature for  at  least  24  hours.  The  glycerol  will  extract  the  pepsinogen. 
Separate,  with  a  pipette  or  by  other  means,  the  glycerol  from  the  pieces 
of  mucous  membrane  and  use  the  glycerol  extract  as  required  in  the 
later  experiments. 

Products  of  Gastric  Digestion. 

Into  the  artificial  gastric  juice,  prepared  as  above  described,  place 
the  protein  material  (fibrin,  coagulated  egg-white,  or  lean  beef)  pro- 
vided for  you  by  the  instructor,  add  0.4  per  cent  hydrochloric  acid  as 
suggested  by  the  instructor  and  keep  the  digestion  mixture  at  40°  C, 
for  2  to  3  days.  Stir  frequently  and  keep  free  hydrochloric  acid  present 
in  the  solution  (for  tests  for  free  hydrochloric  acid  see  p.  131). 

The  original  protein  has  been  digested  and  the  solution  now  con- 
tains the  products  of  peptic  proteolysis,  i.  e.,  acid  metaprotein  (acid 
albuminate),  proteoses,  peptones,  etc.  The  insoluble  residue  may 
include  nuclein  and  anti-albumid.  Filter  the  digestive  mixture  and 
after  testing  ior  free  hydrochloric  acid  neutralize  the  filtrate  with  potas- 
sium hydroxide  solution.  If  any  of  the  acid  metaprotein  (acid  albu- 
minate) is  still  untransformed  into  proteoses  it  will  precipitate  upon  neutral- 
ization. If  any  precipitate  forms  heat  the  mixture  to  boiling,  and  filter. 
If  no  precipitate  forms  proceed  without  filtering. 

We  now  have  a  solution  containing  a  mixture  consisting  princi- 
pally of  proteoses  and  peptones.  Separate  and  identify  the  proteoses 
and  peptones  according  to  the  directions  given  on  pages  120  and  121. 

Tests  for  Free  and  Combined  HCl. 

These  tests  are  made  with  a  class  of  reagents  known  as  indicators, 
so-called  because  they  show  changes  of  color  according  to  the  degree  of 
acidity  (or  alkalinity)  of  the  solution.  They  behave  as  though  they 
were   weak  acids  or  bases   whose  ions  and  unionized  molecules  have 


GASTRIC    DIGESTION.  I31 

different  colors.  Modern  theories  of  color  in  organic  compounds  how- 
ever class  them  as  tautomeric  substances. 

A  neutral  solution  is  one  in  which  there  are  equal  numbers  of  hydro- 
gen and  hydroxyl  ions.  An  acid  solution  has  a  preponderance  of  hydro- 
gen ion  and  an  alkaline  solution  an  excess  of  hydroxyl  ion.  All  indi- 
cators do  not  show  changes  of  color  at  the  true  neutral  point,  but  at 
some  fixed  decree  of  acidity  (or  alkalinity),  i.  e.,  at  a  definite  hydrogen  or 
hydroxyl  ion  concentration.  Those  indicators  which  change  color  at  the 
approximate  true  neutral  point  are  litmus  and  rosolic  acid,  while  phenol- 
phthalein  changes  color  in  a  slightly  alkaline  solution.  Congo  red, 
sodium  alizarin  sulphonate  and  tropaeolin  OO  are  examples  of  indicators 
which  change  color  in  an  acid  solution. 

Organic  acids  are  not  sufficiently  strong,  i.  e.,  do  not  produce  enough 
hydrogen  ion,  to  cause  color  changes  with  the  last-mentioned  class  of 
indicators;  litmus,  rosolic  acid,  and  phenolphthalein  however  indicate 
the  hydrogen  ion  concentration  of  organic  acids  or  their  solutions.  Even 
very  dilute  solutions  of  mineral  acids  are  sufficiently  acid  to  produce 
color  changes  with  congo  red,  etc.  Phenolphthalein,  which  changes 
color  in  a  weakly  alkaline  solution,  is  used  to  indicate  the  presence  of 
acid  combined  with  weakly  alkaline  substances  (as  protein),  as  well  as 
the  other  types  of  acid  and,  hence,  is  used  to  indicate  the  total  acidity. 
The  differentiation  between  the  various  forms  of  acidity  depends  upon 
the  above  facts. 

The  hydrogen  ion  concentrations  at  which  some  common  indicators 
show  the  most  characteristic  change  of  color  are  given  below.  Concen- 
trations are  expressed  in  approximate  moles  of  hydrogen  ion  per  liter. 

True  nature 
Indicator.  Hydrogen  ion  of  solution 

concentration.  when  the 

color  changes. 

Rosolic  acid i  X  io~"' Neutral. 

Litmus Between  i  X  lo"*^  and  i  X  io"~' . .   Neutral. 

Tropa-olin  OO i  X  io~^ Acid. 

Dimethyl-amino-azobenzene  ..   Between  i  X  io~' and  iXio~*..   .\cid. 
Sodium  alizarin  sulphonate. . . .   Between  i  X  lO""*  and  i  X  io~*. .  Acid. 

Congo  red Between  i  X  io~°  and  i  X  10—^ . .  Acid. 

Phenolphthalein Between  i  X  lO""'  and  i  X  lo""'. .  Alkaline. 

Examine  each  of  the  following  solutions  by  means  of  the  tests  given 
below  and  report  the  results  in  a  form  similar  to  the  chart  given  on 
page  133:  (i)  0.2  per  cent  free  hydrochloric  acid.  (2)  0.05  per  cent 
free  hydrochloric  acid.  (3)  0.0 1  per  cent  free  hydrochloric  acid.  (4) 
0.05  per  cent  combined  hydrochloric  acid  (see  p.  126).  (5)  i  per  cent 
lactic  acid.  (6)  Equal  volumes  of  0.2  per  cent  free  hydrochloric  acid  and 
I  per  cent  lactic  acid.     (7)  i  per  cent  potassium  hydroxide. 


132  PHYSIOLOGICAL    CHEMISTRY. 

1.  Dimethyl-amino-azobenzene    (or   Topfer's   Reagent),^ 

N(CH3),-C,H,-N^N-C,H,. 

Place  1-2  drops  of  the  reagent  in  the  solution  to  be  tested.  Free  min- 
eral acid  (hydrochloric  acid)  is  indicated  by  the  production  of  a  pinkish- 
red  color.     If  free  acid  is  absent  a  yellow  color  ordinarily  results. 

2.  Giinzberg's  Reagent.^ — ^Place  1-2  drops  of  the  reagent  in  a 
small  porcelain  evaporating  dish  and  carefully  evaporate  to  dryness 
over  a  low  flame.  Insert  a  glass  stirring  rod  into  the  mixture  to  be 
tested  and  draw  the  moist  end  of  the  rod  through  the  dried  reagent. 
Warm  again  gently  and  note  the  production  of  a  purplish-red  color 
in  the  presence  oifree  hydrochloric  acid. 

3.  Boas'  Reagent.^ — Perform  this  test  in  the  same  manner  as 
2,  above.  Free  hydrochloric  acid  is  indicated  by  the  production  of 
a  rose-red  color  which  becomes  less  pronounced  on  cooling. 

4.  Congo  Red,* 
NH2  •  SOgNa 


SOgNa  NH2 

Conduct  this  test  according  to  the  directions  given  under  i  or  2,  above 
A  blue  color  indicates  free  hydrochloric  acid,  a  violet  color  indi- 
cates an  organic  acid  and  a  brown  color  indicates  combined  hydro- 
chloric acid.  Congo-red  paper,  made  by  immersing  ordinary  fiilter 
paper  in  the  indicator  and  subsequently  drying,  may  be  used  in  this 
test. 

5.  Tropaeolin  00/ 

NH(CeH5)  -CgH,  -N  =N  -C,H,  -SOgNa. 

Place  2  drops  of  the  solution  to  be  tested  and  i  drop  of  the  indicator 
in  an  evaporating  dish  and  evaporate  to  dryness  over  a  low  fiame.  The 
formation  of  a  reddish-violet  color  indicates /r^e  hydrochloric  acid. 

This  test  may  also  be  conducted  in  the  same  manner  as  2, 
above. 

'  To  prepare  Topfer's  reagent  dissolve  0.5  gram  of  climethyl-amino-azobenzene  in  100  c.c. 
of  95  per  cent  alcohol. 

*  Giinzberg's  reagent  is  prepared  by  dissolving  2  grams  of  phloroglucinol  and  i  gram  of 
vanillin  in  100  c.c.  of  95  per  cent  alcohol. 

■^  Boas'  reagent  is  prepared  by  dissolving  5  grams  of  resorcinol  and  3  grams  of  sucrose  in 
100  c.c.  of  50  per  cent  alcohol. 

'  This  indicator  is  prepared  by  dissolving  0.5  gram  of  congo  red  in  90  c.c.  of  water  and 
adding  10  c.c.  of  95  per  cent  alcohol. 

^Prepared  by  dissolving  0.05  gram  of  tropa;olin  OO  in  100  c.c.  of  50  per  cent  alcohol. 


GASTRIC    DIGESTION. 


133 


6.  Phenolphthalein/ 


C„H,OH 


C— C«H,OH 


C„H, 


O 


\ 

o 

Add  the  indicator  directly  to  the  solution,  or  apply  the  test  according  to 
the  directions  given  under  2  on  page  134.  This  indicator  serves  to 
denote  the  total  acidity  since  it  is  acted  upon  by  free  mineral  acids, 
combined  acids,  organic  acids,  and  acid  salts.  A  red  color  indicates  the 
presence  of  an  alkali  and  the  indicator  is  colorless  in  the  presence  of  a 
neutral  or  acid  reaction.  This  indicator  is  unsatisfactory  in  the  pres- 
ence of  ammonia. 


7.  Sodium  Alizarin  Sulphonate,^ 

CO 


(OH), 


C„H 


C„H 


CO  S03Na 

This  indicator  may  be  used  directly  in  the  solution  to  be  tested,  or  the 
test  may  be  applied  as  2,  page  134.  It  serves  to  indicate  all  acid  re- 
actions except  those  due  to  combined  acids.  A  reddish-violet  color 
indicates  an  alkaline  reaction,  while  a  yellow  color  indicates  an  acid 
reaction  due  to  a  free  mineral  acid,  an  organic  acid,  or  an  acid  salt. 
Report  the  results  of  your  tests  tabulated  in  the  form  given  below: 


Solutions  Examined. 


Name  of  Indicator. 


0.2    ■o 

HCl. 


0.05  % 
HCl. 


O.OI    .0 

HCl. 


0.0s  %  I  7c 

Combined     Lactic 
HCl.  Acid. 


Equal  Vols. 
0.2%  HCl  1^0 

and  I  %       '    KOH. 
Lactic  Acid,  i 


Topfer's  Reagent. 

1 

Giinzberg's  Reagent.           j 

Boas'  Reagent.                    | 

Congo  Red. 

Tropaeolin  GO.                       '                                      j 

Phenolphthalein. 

Ali7.arin.                                 [ 

'  This  indicator  is  prepared  by  dissolving  i  gram  of  phenolphthalein  in  100  c.c.  of  95 
per  cent  alcohol. 

-  Prepare  this  indicator  by  dissolving  i  gram  of  sodium  alizarin  sulphonate  in  100  c.c. 
of  water. 


134  PHYSIOLOGICAL    CHEMISTRY. 

GENERAL  EXPERIMENTS  ON  GASTRIC  DIGESTION. 

1.  Conditions  Essential  for  the  Action  of  Pepsin. — ^Prepare  four 
test-tubes  as  follows: 

(a)  Five  c.c.  of  pepsion  solution. 

(b)  Five  c.c.  of  0.4  per  cent  hydrochloric  acid. 

(c)  Five  c.c.  of  pepsin-hydrochloric  acid  solution. 

{d)  Two  or  three  c.c.  of  pepsin  solution  and  2-3  c.c.  of  0.5  per  cent 
sodium  carbonate  solution. 

Introduce  into  each  tube  a  small  piece  of  fibrin  and  place  them  in  the 
incubator  or  water-bath  at  40°  C.  for  one-half  hour,  carefully  noting  any 
changes  which  occur.  ^  Now  combine  the  contents  of  tubes  (a)  and 
(b)  and  see  if  any  further  change  occurs  after  standing  at  40°  C.  for 
15-20  minutes.  Explain  the  results  obtained  from  these  five  experi- 
ments. 

2.  Influence  of  Different  Temperatures. — In  each  of  four  test- 
tubes  place  5  c.c.  of  pepsin-hydrochloric  acid  solution.  Immerse  one 
tube  in  cold  water  from  the  faucet,  keep  a  second  tube  at  room  tem- 
perature and  place  a  third  in  the  incubator  or  water-bath  at  40°  C.  Boil 
the  contents  of  the  fourth  tube  for  a  few  moments,  then  cool  and  also 
keep  it  at  40°  C.  Into  each  tube  introduce  a  small  piece  of  fibrin  and 
note  the  progress  of  digestion.  In  which  tube  does  the  most  rapid 
digestion  occur  ?     Explain  this. 

3.  The  Most  Favorable  Acidity. — Prepare  three  tubes  as  follows: 
(a)  Five  c.c.  of  0.2  per  cent  pepsin-hydrochloric  acid  solution. 

{b)  Two  or  three  c.c.  of  0.2  per  cent  hydrochloric  acid  +  i  c.c.  of 
concentrated  hydrochloric  acid  +  5  c.c.  of  pepsin  solution. 

(c)  One  c.c.  of  0.2  per  cent  pepsin-hydrochloric  acid  solution  -f  5  c.c. 
of  water. 

Introduce  a  small  piece  of  fibrin  into  each  tube,  keep  them  at  40° 
C,  and  note  the  progress  of  digestion.  In  which  degree  of  acidity 
does  the  fibrin  digest  the  most  rapidly  ? 

4.  Differentiation  Between  Pepsin  and  Pepsinogen. — Prepare 
five  tubes  as  follows: 

(a)  Few  drops  of  glycerol  extract  of  pepsinogen  -\-  2-3  c.c.  of  water. 

(b)  Few  drops  of  glycerol  extract  of  pepsinogen  -1-  5  c.c.  of  0.2  per 
cent  hydrochloric  acid. 

'  Digestion  of  fibrin  in  a  pepsin-hydrochloric  acid  solution  is  indicated  first  by  a  swelling 
of  the  protein  due  to  the  action  of  the  acid,  and  later  by  a  disintegration  and  dissolving  of 
the  fibrin  due  to  the  action  of  the  pepsin-hydrochloric  acid.  If  uncertain  at  any  time  whether 
digestion  has  taken  place,  the  solution  under  examination  may  be  filtered  and  the  biuret 
test  ajjj>lied  to  the  filtrate.  A  positive  reaction  will  signify  the  presence  of  acid  metaprotein 
(acid  albuminate;,  proteoses  (albumoses),  or  peptones,  the  presence  of  any  one  of  which 
would  indicate  that  digestion  has  taken  place. 


GASTRIC   DIGESTION.  I35 

(c)  Few  drops  of  glycerol  extract  of  pepsinogen  +  5  c.c.  of  0.5  per 
cent  sodium  carbonate. 

(d)  Two  or  three  c.c.  of  pepsin  solution  +  2-3  c.c.  of  i  per  cent 
sodium  carbonate. 

(e)  Few  drops  of  glycerol  extract  of  pepsinogen  +  5  c.c.  of  i  per 
cent  sodium  carbonate. 

Add  a  small  piece  of  fibrin  to  the  contents  of  each  tube,  keep  the 
live  tubes  at  46°  C.  for  one-half  hour  and  observe  any  changes  which 
may  have  occurred.  To  (a)  add  an  ec[ual  volume  of  0.4  per  cent  hydro- 
chloric acid,  neutralize  (c),  (d)  and  (e)  with  hydrochloric  acid  and  add 
an  equal  volume  of  0.4  per  cent  hydrochloric  acid.  Place  these  tubes 
at  40°  C.  again  and  note  any  further  changes  which  may  occur.  What 
contrast  do  we  find  in  the  results  from  the  last  three  tubes  T-*  Why  is 
this  so  ? 

5.  Comparative  Digestive  Power  of  Pepsin  with  Different 
Acids. — Prepare  a  series  of  tubes  each  containing  one  of  the  following 
acids:  0.5  per  cent  acetic,  lactic,  oxalic,  salicylic,  tannic,  and  butyric, 
and  0.2  per  cent  hydrochloric,  sulphuric,  nitric,  arscnious,  and  com- 
bined hydrochloric.  To  each  acid  add  a  few  drops  of  the  glycerol  extract 
of  pig's  stomach  and  a  small  piece  of  fibrin.  Shake  well,  place  at  40° 
C,  and  note  the  progress  of  digestion.  In  which  tubes  does  the  most 
rapid  digestion  occur  ? 

6.  Influence  of  Metallic  Salts,  etc. — ^Prepare  a  series  of  tubes 
and  into  each  tube  introduce  4  c.c.  of  pepsin-hydrochloric  acid  solu- 
tion and  1/2  c.c.  of  one  of  the  chemicals  listed  in  Experiment  18  under 
Salivary  Digestion,  page  66.  Introduce  a  small  piece  of  fibrin  into 
each  of  the  tubes  and  keep  them  at  40°  C.  for  one-half  hour.  Note 
the  variations  in  the  progress  of  digestion.  Where  has  the  least  rapid 
digestion  occurred  ? 

7.  Sahli's  Desmoid  Reaction. — This  is  a  method  for  testing  gastric 
function  without  using  the  stomach  tube.  The  underlying  principle 
of  the  test  is  the  fact  that  raw  catgut  may  be  digested  in  gastric  juice 
but  is  entirely  indigestible  in  pancreatic  juice.  The  test  is  made  as 
follows:  A  methylene-blue  pill  is  introduced  into  a  small  rubber  bag 
and  the  mouth  of  the  bag  subsequently  tied  with  catgut.^  The  small 
bag  is  then  ingested  immediately  after  the  mid-day  meal  and  the  urine 
examined  5,  7,  9  and  18-20  hours  later  for  methylene  blue.     If  methylene 

'  About  0.05  gram  of  methylene  blue  is  mixed  with  sufficient  ext.  glycyrrhiza  to  form 
a  pill  about  3-4  mm.  in  diameter.  The  pill  is  then  placed  in  the  center  of  a  square  piece  of 
thin  rubber  dam  and  a  little  bag-like  receptacle  constructed  by  a  twisting  movement.  The 
neck  of  the  bag  is  then  closed  by  wrapping  three  turns  of  catgut  about  it.  The  most  satis- 
factory catgut  to  use  is  number  00  raw  catgut  which  has  previously  been  soaked  in  water 
until  soft.  When  ready  for  use  the  bag  should  sink  instantly  when  placed  in  water  and 
be  water-tight. 


136  PHYSIOLOGICAL    CHEMISTRY. 

blue  is  present  in  appreciable  qaantity,  it  will  impart  to  the  urine  a 
greenish-blue  color.  If  not  present  in  sufficient  amount  to  impart  this 
color  the  urine  should  be  boiled  with  1/5  its  volume  of  glacial  acetic 
acid,  w^hereupona  gieenish-bluecolor  results  if  thechromogenof  methylene 
blue  is  present.  This  contingency  seldom  arises,  however,  inasmuch 
as  in  most  cases  of  uncolored  urine  it  will  be  found  that  the  rubber  bag 
has  passed  through  the  stomach  unopened.  If  the  methylene  blue  is 
found  in  the  urine  inside  of  18-20  hours  a  satisfactory  gastric  function 
is  indicated. 

For  Einhorn's  bead  method  for  the  study  of  digestive  function,  see 
chapter  on  Feces. 

8.  Testing  the  Motor  and  Functional  Activities  of  the  Stomach. 
— This  test  is  performed  the  same  as  Experiment  19  under  Salivary 
Digestion,  page  67.  If  the  experiment  was  carried  out  under  salivary 
digestion  it  will  not  be  necessary  to  repeat  it  here. 

9.  Influence  of  Bile. — ^Prepare  five  tubes  as  follows: 

(a)  Five  c.c.  of  pepsin-hydrochloric  acid  solution  +  1/2-1  c.c.  of 
bile. 

(b)  Five  c.c.  of.  pepsin-hydrochloric  acid  solution  +  1-2  c.c.  of 
bile. 

(c)  Five  c.c.  of  pepsin-hydrochloric  acid  solution  +  2-3  c.c.  of  bile. 

(d)  Five  c.c.  of  pepsin-hydrochloric  acid  solution  +  5  c.c.  of  bile. 

(e)  Five  c.c.  of  pepsin-hydrochloric  acid  solution. 

Introduce  into  each  tube  a  small  piece  of  fibrin.  Keep  the  tubes 
at  40°  C.  and  note  the  progress  of  digestion.  Does  the  bile  exert  any 
appreciable  influence  ?     How  ? 

10.  Influence  of  Gastric  Rennin  on  Milk. — Prepare  a  series  of 
five  tubes  as  follows: 

(a)  Five  c.c.  of  fresh  milk  +  0.2  per  cent  hydrochloric  acid  (add 
slowly  until  precipitate  forms). 

{b)  Five  c.c.  of  fresh  milk  -|-  5  drops  of  rennin  solution. 

(c)  Five  c.c.  of  fresh  milk  +  10  drops  of  0.5  per  cent  sodium  car- 
bonate solution. 

(d)  Five  c.c.  of  fresh  milk  +  10  drops  of  a  saturated  solution  of 
ammonium  oxalate. 

(e)  Five  c.c.  of  fresh  milk  -h  5  drops  of  0.2  per  cent  hydrochloric  acid. 
Now  to  each  of  the  tubes  (c),  (d),  and  (e)  add  5  drops  of  rennin  solution. 
Place  the  whole  series  of  five  tubes  at  40°  C.  and  after  10-15  minutes 
note  what  is  occurring  in  the  diflerent  tubes.  Give  a  reason  for  each 
particular  result. 

11.  Tests  for  Lactic  Acid. — (a)  Ufelmann's  Reaction. — To  a  small 


GASTRIC    DIGESTION.  1 37 

quantity  of  UlTelmann's  reagent'  in  a  test-tube  add  a  few  drops 
of  a  lactic  acid  solution.  The  amethyst-blue  color  of  the  reagent  is 
displaced  by  a  straw  yellow.  Other  organic  acids  gi\'e  a  similar  reaction. 
Mineral  acids  such  as  hydrochloric  acid  discharge  the  blue  coloration 
lea%ing  a  colorless  solution.  In  other  words,  the  color  of  the  reagent 
is  weakened  in  the  presence  of  an  acid  reaction. 

(b)  Ferric  Chloride  Test. — Place  lo  c.c.  of  very  dilute  ferric  chloride 
in  each  of  five  tubes.  To  the  first  add  2  c.c.  of  0.2  per  cent  hydro- 
chloric acid,  to  the  second  2  c.c.  of  10  per  cent  alcohol,  to  the  third 
2  c.c.  of  2  per  cent  sucrose,  to  the  fourth  2  c.c.  of  lactic  acid  and  to  the 
fifth  2  c.c.  of  peptone  solution. 

It  is  evident  from  the  results  obtained  that  neither  of  the  tests  given 
above  is  satisfactory  for  the  detection  of  lactic  acid  in  the  presence  of 
other  substances  such  as  we  find  in  the  gastric  contents. 

A  satisfactory  deduction  regarding  the  presence  of  lactic  acid  can 
only  be  made  after  extracting  the  gastric  contents  with  ether,  evapora- 
ting the  ether  extract  to  dryness,  and  dissolving  the  residue  in  water. 
This  residue  will  not  contain  any  of  the  contaminations  which  interfere 
with  the  simple  tests  as  tried  above,  and  therefore  if  either  of  the  tests 
is  now  tried  on  the  dissolved  residue  of  the  ether  extract  we  may  form 
an  accurate  conclusion  regarding  the  presence  of  lactic  acid. 

(c)  Hopkins^  Thiophenc  Reaction.- — Place  about  5  c.c.  of  concen- 
trated sulphuric  acid  in  a  test-tube  and  add  one  drop  of  a  saturated  solu- 
tion of  copper  sulphate."  Introduce  a  few  drops  of  the  solution  to  be 
tested,  shake  the  tube  well,  and  immerse  it  in  the  boiling  water  of  a 
beaker-water-bath  for  one  or  two  minutes.  Now  remove  the  tube,  cool 
it  under  running  water,  add  2-3  drops  of  a  dilute  alcoholic  solution^  of 
thiophene,  C^H^S,  from  a  pipette,  replace  the  tube  in  the  beaker  and 
carefully  observe  any  color  change  which  may  occur.  Lactic  acid  is 
indicated  by  the  appearance  of  a  bright  cherry-red  color  which  forms 
rapidly.  This  color  may  be  made  more  or  less  permanent  by  cooling 
the  tube  as  soon  as  the  color  is  produced.  Excess  of  thiophene  produces 
a  deep  yellow  or  brown  color  with  sulphuric  acid.  The  test  is  not 
wholly  specific  though  the  author  claims  it  to  be  more  so  than  Uffelmann's 
reaction. 

12.  Qualitative  Analysis  of  Stomach  Contents. — Take  100  c.c. 
of  stomach  contents  and  analyze  it  according  to  the  following  scheme: 

■  Uffelmann's  reagent  is  prepared  by  adding  ferric  chloride  solution  to  a  i  per  cent 
solution  of  carbolic  acid  until  an  amethyst-blue  color  is  obtained,  due  to  the  formation  of  a 
ferric  salt  of  carbolic  acid. 

-  This  is  added  to  catalyze  the  oxidation  which  follows. 

'  About  10-20  drops  in  loo  c.c.  of  95  per  cent  alcohol. 


138  PHYSIOLOGICAL   CHEMISTRY. 

Stomach  Contents. 
Filter  and  test  the  filtrate  for  free  hydrochloric  acid. 


I  I 

Filtrate  I.  Residue. 

Divide  into  two  parts.  Discard  after  making  a  microscopical  exami- 
I  nation. 


I  I 

Filtrate  II.  Filtrate  III. 
One-fifth  portion.                                  Four-fifths  portion. 

Test  for:  Neutralize  carefully;  any  precipitate  is  acid  meta- 

(a)  Pepsin.  protein  (acid  albuminate).     If  a  precipitate  forms 

(b)  Bile  (see  page  162).  filter  and  divide  the  filtrate  into  two  parts.     If  no 

(c)  Starch.  precipitate  forms  divide  the  solution  into  two  parts 

(d)  Dextrin.  without  filtering. 


Filtrate  IV.  Filtrate  V. 

Two-thirds  portion.  One-third  portion. 

Heat    to    boiling    to    remove    coagulable 
proteins.     If  any  precipitate  forms  filter 

it  off;  if  there  is  no  precipitate  proceed  Test  for: 

directly  with  the  tests.  (a)  Lactic  acid. 

Test  for:  (b)   Gastric  rennin. 

(a)  Sugar.     (Differentiate  between  the  various  (c)   Salivary  amylase 
sugars  by  the  use  of  the  scheme  on  page 

5S.) 

(b)  Proteoses. 

(c)  Peptones. 


CHAPTER  Vn. 

FATS. 

Fats  occur  very  widely  distributed  in  the  plant  and  animal  king- 
doms, and  constitute  the  third  general  class  of  food  stuffs.  In  plant 
organisms  they  are  to  be  found  in  the  seeds,  roots,  and  fruit  while  each 
individual  tissue  and  organ  of  an  animal  organism  contains  more  or 
less  of  the  substance.  In  the  animal  organism  fats  are  especially  abundant 
in  the  bone  marrow  and  adipose  tissue.  They  contain  the  same  elements 
as  the  carbohydrates,  i.  e.,  carbon,  hydrogen,  and  oxygen,  but  the  oxygen 
is  present  in  smaller  percentage  than  in  the  carbohydrates  and  the 
hydrogen  and  oxygen  are  not  present  in  the  proportion  to  form  water. 


Fig.  36. — Beef  Fat.     {Long.) 

Chemically  considered  the  fats  are  esters*  of  the  tri-atomic  alcohol, 
glycerol,  and  the  mono-basic  fatty  acids.  In  the  formation  of  these 
fats  three  molecules  of  water  result.  This  water  may  arise  in  either 
of  two  ways.  First,  by  the  replacement  of  the  H  of  each  of  the  OH 
groups  of  glycerol  by  a  fatty  acid  radical,  giving  the  following  formula 
in  which  R,  R'  and  R"  represent  fatty  acid  radicals, 

CH3OR 

CHOR' 

I 
CH2OR" 

'  .\n  ester  is  an  oxyacid,  one  of  whose  acid  hydrogens  is  replaced  by  an  organic  radical 


I40  PHYSIOLOGICAL    CHEMISTRY. 

Second,  by  the  replacement  of  the  H's  of  the  carboxyl  groups  of  the 
three  fatty  acid  molecules  by  the  glycerol  radical,  thus  yielding  the 
following  type  of  formula  in  which  R  represents  the  glycerol  radical, 

OOCH3,C,, 

R-00CH3,C, 

\ 

OOCH3,C,, 

Of  these  two  processes  the  second  is  the  more  logical  procedure  from 
the  standpoint  of  the  ionic  theory.  The  three  fatty  acid  radicals  entering 
into  the  structure  of  a  neutral  fat  may  be  the  radicals  of  the  same  fatty 
acid  or  they  may  consist  of  the  radicals  of  three  different  fatty  acids. 

By  hydrolysis  of  a  neutral  fat,  i.  e.,  by  the  addition  to  the  molecule  of 
those  elements  which  are  eliminated  in  the  formation  of  the  fat  from 
glycerol  and  fatty  acid,  it  may  be  resolved  into  its  component  parts,  /'.  e., 
glycerol  and  fatty  acid.  In  the  case  of  palmitin  the  following  would 
be  the  reaction: 

C3H,(0-C„H3,CO)3  +  3H,0-^C3H,(OH)3  +  3(C,,H3,COOH). 

Palmitin.  Glycerol-  Palmitic  acid- 

This  process  is  called  saponification  and  may  be  produced  by  boiling 
with  alkalis;  by  the  action  of  steam  under  pressure;  by  long-continued 
contact  with  air  and  light;  by  the  action  of  certain  bacteria  and  by  fat- 
splitting  enzymes  or  lipases,  e.  g.,  pancreatic  lipase  (see  page  151).  The 
cells  forming  the  walls  of  the  intestines  evidently  possess  the  peculiar 
property  of  synthesizing  the  glycerol  and  fatty  acid  thus  formed  so  that 
after  absorption  these  bodies  appear  in  the  blood  not  in  their  individual 
form  but  as  neutral  fats.  This  synthesis  is  similar  to  that  enacted  in 
the  absorption  of  protein  material  where  the  peptones  are  synthesized 
into  albumin  in  the  act  of  absorption. 

The  principal  animal  fats  with  which  we  have  to  deal  are  stearin,  palm- 
itin, olein,  and  butyrin.  Such  less  important  forms  as  laurin  and  myristin 
may  occur  abundantly  in  plant  organisms.  The  older  system  of  nomencla- 
ture for  these  fats  was  to  apply  the  prefix  "tri"  in  each  case  {e.  g.,  tri- 
palmitin)  since  there  fatty  acid  radicals  are  contained  in  the  neutral 
fat  molecule. 

Fats  occur  ordinarily  as  mixtures  of  several  individual  fats.  For 
example,  the  fat  found  in  animal  tissues  is  a  mixture  of  olein,  palmitin 
and  stearin,  the  percentage  of  any  one  of  these  fats  present  depending 
upon  the  particular  species  of  animal  from  whose  tissue  the  fat  was 
derived.  Thus  the  ordinary  mutton  fat  contains  more  stearin  and  less 
olein  than  the  pork  fat.     Human  fat  contains  from  67  per  cent  to  85  per 


FATS.  141 

cent  of  olcin  and  according  to  Hcncdict  and  Osterbcrg,  upon  analysis 
yields  76.08  per  cent  of  carbon  and  11.78  per  cent  of  hydrogen.  Butter 
consists  in  large  part  of  olein  and  palmitin.  Stearin,  butyrin,  caproin 
and  traces  of  other  fats  are  also  present. 

Pure  neutral  fats  are  odorless,  tasteless,  and  generally  colorless. 
Thev  arc  insoluble  in  the  ordinary  protein  solvents  such  as  water,  salt 
solutions,  and  dilute  acids  and  alkalis,  but  are  very  readily  soluble  in  ether, 
benzene,  chloroform,  and  boiling  alcohol.  The  neutral  fats  are  non- 
volatile substances  possessing  a  neutral  reaction.  If  allowed  to  remain 
in  contact  with  the  air  for  a  sufficient  length  of  time  they  become  yellow 
in  color,  assume  an  acid  reaction  and  are  said  to  be  rancid.  The  neutral 
fats  may  be  crystallized,  some  of  them  with  great  facility.  The  crystalline 
forms  of  some  of  the  more  common  fats  arc  reproduced  in  Figs.  36,  37 
and  38  on  pages  139, 142  and  144.  Each  individual  fat  possesses  a  specific 
melting-  or  boiling-point  (according  to  whether  the  body  is  solid  or  fluid  in 
character)  and  this  property  of  melting  or  boiling  at  a  definite  temperature 
may  be  used  as  a  means  of  differentiation  in  the  same  way  as  the  coag- 
ulation temperature  (see  page  117)  is  used  for  the  dillferentiation  of  coag- 
ulable  proteins.  When  shaken  with  water,  or  a  solution  of  albumin, 
soap,  or  acacia,  the  liquid  fats  are  finely  divided  and  assume  a  condition 
known  as  an  emulsion.  The  emulsion  with  water  is  transitory,  while  the 
emulsions  with  soap,  acacia,  or  albumin,  are  permanent. 

The  fat  ingested  continues  essentially  unaltered  until  it  reaches  the  in- 
testine where  it  is  acted  upon  by  pancreatic  lipase  (steapsin)  the  fat-split- 
ting enzyme  of  the  pancreatic  juice  (see  page  151),  and  glycerol  and  fatty 
acid  are  formed  from  a  large  portion  of  the  fat.  Part  of  the  fatty  acid 
thus  formed  is  dissolved  in  the  bile  and  absorbed  while  the  remainder 
unites  with  the  alkalis  of  the  pancreatic  juice  and  forms  soluble  soaps. 
These  soaps  may  further  act  to  produce  an  emulsion  of  the  remaining 
fat  and  thus  aid  in  its  absorption.  That  bile  is  of  assistance  in  the  absorp- 
tion of  fat  is  indicated  by  the  increase  of  fat  in  the  feces  when  for  any 
reason  bile  does  not  pass  into  the  intestine.  That  fat  is  not  absorbed 
unsplit  in  the  form  of  an  emulsion  has  recently  been  redemonstrated  by 
Whitehead  *  in  a  histological  study  of  the  absorption  in  the  cat's  intestine  of 
fat  stained  with  Sudan  III.  Whitehead  considers  that  fat  was  not 
absorbed  unsplit  because  no  dye  was  jound  in  the  lacteals.  Mendel" 
has  pointed  out  that  Sudan  III  is  soluble  in  fatty  acids  as  well  as  fats, 
and  therefore  its  presence  in  the  lacteals  furnishes  no  evidence  "  for  or 
against  the  possibility  of  the  absorption  of  fats  prior  to  their  digestion." 
The  failure  to  find  Sudan  III  in  the  lacteals  may  have  been  due  to  the 

*  Whitehead:  American  Journal  of  Physiology,  24,  294,  1909. 

*  Mendel:  Ibid.,  p.  493, 


142  PHYSIOLOGICAL    CHEMISTRY. 

fact  that  in  postmortem    examinations    these   vessels    are    often    found 
collapsed  and  empty. 

The  fat  distributed  throughout  the  animal  body  is  formed  partly 
from  the  ingested  fat  and  partly  from  carbohydrates  and  the  "carbon 
mioety"  of  protein  material.  The  formation  of  adipocere  and  the 
occurrence  oi  fatty  degeneration  are  sometimes  given  as  proofs  of  the 
formation  of  fat  from  protein.  This  is  questioned  by  many  investigators. 
Rather  more  satisfactory  and  direct  proof  of  the  formation  of  fat  from 
protein  material  has  been  obtained  by  Hofmann  in  experimentation 


Fig.  37. — Mutton  Fat.     {Long.) 

with  fly-maggots.  The  normal  content  of  fat  in  a  number  of  maggots 
was  determined  and  later  the  fat  content  of  others  which  haddeveloped  in 
blood  (84  per  cent  of  the  solid  matter  of  blood  plasma  is  protein  material) 
was  determined.  The  fat  content  was  found  to  have  increased  700  to 
1 100  per  cent  as  a  result  of  the  diet  of  blood  proteins.  The  celebrated 
experiments  of  Pettenkofer  and  Voit,  however,  have  furnished  what  is, 
perhaps,  the  most  substantial  positive  evidence  of  the  formation  of 
fat  from  protein.  These  investigators  fed  dogs  large  amounts  of  lean 
meat,  daily,  and  through  subsequent  urinary  and  fecal  examinations 
were  enabled  to  account  for  only  part  of  the  ingested  carbon,  although 
obtaining  a  satisfactory  nitrogen  balance.  The  discrepancy  in  the  carbon 
balance  was  explained  upon  the  theory  that  the  protein  of  the  ingested 
meat  had  been  split  into  a  nitrogenous  and  a  non-nitrogenous  portion  in 
the  organism,  and  that  the  non-nitrogenous  portion,  the  so-called  "carbon 
moiety"  of  the  protein,  had  been  subsequently  transformed  into  fat  and 
deposited  as  such  in  the  tissues  of  the  organism.  Some  investigators  are 
not  inclined  to  accept  these  data  regarding  the  formation  of  fat  from 
protein  as  conclusive. 


FATS.  143 

Later  evidence  in  favor  of  the  formation  of  fat  from  protein  has 
been  furnished  by  the  experiments  of  Weinland.  This  investigator 
worked  with  the  larvae  of  Calliphora,^  these  larvae  being  rubbed  up 
in  a  mortar^  with  Witte's  peptone  and  water  to  form  a  homogeneous 
mixture.  After  placing  these  mixtures  at  38°  C.  for  24  hours  the  fat 
content  was  found  to  have  increased,  as  much  as  140  per  cent  in  some 
instances.  The  active  agency  in  this  transformation  of  fat  is  the  larval 
tissue  since  the?  tissues  of  both  the  dead  and  li\'ing  larvae  possess  the 
property.  Data  are  given  from  control  tests  which  show  that  the  action 
of  bacteria  in  this  transformation  of  protein  was  excluded. 

Experiments  on  Fats. 

1.  Solubility. — Test  the  solubility  of  olive  oil  in  each  of  the  ordi- 
nary solvents  (see  page  27)  and  in  cold  alcohol,  hot  alcohol,  chloroform, 
ether,  and  carbon  tetrachloride. 

2.  Formation  of  a  Transparent  Spot  on  Paper. — Place  a  drop 
of  olive  oil  upon  a  piece  of  ordinary  writing  paper.  Note  the  trans- 
parent appearance  of  the  paper  at  the  point  of  contract  with  the  fat. 

3.  Reaction. — Try  the  reaction  oi  fresh  olive  oil  to  litmus,  congo  red 
and  phenolphthalein.  Repeat  the  test  with  rancid  olive  oil.  WTiat  is 
the  reaction  of  a  fresh  fat  and  how  does  this  reaction  change  upon 
allowing  the  fat  to  stand  for  some  time? 

4.  Formation  of  Acrolein.^ — To  a  little  olive  oil  in  a  mortar  add 
some  dry  potassium  bisulphate,  KHSO^,  and  rub  up  thoroughly.  Trans- 
fer to  a  dry  test-tube  and  cautiously  heat.  Note  the  irritating  odor  of 
acrolein.  The  glycerol  of  the  fat  has  been  dehydrolyzed  and  acrylic 
aldehyde  or  acrolein  has  been  produced.  This  is  the  reaction  which 
takes  place: 

CH^OH  CHO 

I  I 

CHOH     -^     CH+2H2O. 

I  II 

CH^OH  CH2 

Glycerol.  Acrolein. 

5.  Emulsification. — (a)  Shake  up  a  drop  of  neutral^  olive  oil  with 
a  little  water  in  a  test-tube.  The  fat  becomes  finely  divided,  forming 
an  emulsion.  This  is  not  a  permanent  emulsion  since  the  fat  separates 
and  rises  to  the  top  upon  standing. 

'  The  ordinary  "blow-fly." 

^  Intact  larva;  were  used  in  some  experiments. 

'  Neutral  olive  oil  may  be  prepared  by  shaking  ordinary  olive  oil  with  a  lo  per  cent  solution 
of  sodium  carbonate.  This  mixture  should  then  be  extracted  with  ether  and  the  ether 
removed  by  evaporation.     The  residue  is  neutral  olive  oil. 


144 


PHYSIOLOGICAL    CHEMISTRY. 


(b)  To  5  c.c.  of  water  in  a  test-tube  add  2  or  3  drops  of  0.5  per  cent 
NaoCOg.  Introduce  into  this  faintly  alkaline  solution  a  drop  of  neutral 
olive  oil  and  shake.  The  emulsion  while  not  permanent  is  not  so  transi- 
tory as  in  the  case  of  water  free  from  sodium  carbonate. 

(c)  Repeat  {b)  using  rancid  olive  oil.  What  sort  .of  an  emulsion 
do  you  get  and  why  ? 

{d)  Shake  a  drop  of  neutral  olive  oil  with  dilute  albumin  solution. 
\^^lat  is  the  nature  of  this  emulsion  ?     Examine  it  under  the  microscope. 

6.  Fat  Crystals. — Dissolve  a  small  piece  of  lard  in  ether  in  a  test- 
tube,  add  an  equal  volume  of  alcohol  and  allow  the  alcohol-ether  mixture 


Fig.  t,S. — Pork  Fat. 


to  evaporate  spontaneously.  Examine  the  crystals  under  the  microscope 
and  compare  them  with  those  reproduced  in  Figs.  36,  37  and  38,  on  pages 
39,  142  and  144. 

7.  Saponification  of  Bayberry  Tallow.^ — ^Fill  a  large  casserole 
two-thirds  full  of  water  rendered  strongly  alkaline  with  solid  potassium 
hydroxide  (a  stick  one  inch  in  length).  Add  about  10  grams  of  bay- 
berry  tallow  and  boil,  keeping  the  volume  constant  by  adding  water  as 
needed.  When  saponification  is  complete"  remove  25  c.c.  of  the  soap 
solution  for  use  in  Experiment  8  and  add  concentrated  hydrochloric 
acid  slowly  to  the  remainder  until  no  further  precipitate  is  produced.^ 
Cool  the  solution  and  the  precipitate  of  free  fatty  acid  will  rise  to  the  sur- 
face and  form  a  cake.     In  this  instance  the  fatty  acid  is  principally  pal- 

'  Baybern-  tallow  is  flerivcd  from  the  fatty  covering  of  the  berries  of  the  wax  myrtle.  It 
s  therefore  frequently  (ailed  "myrtle  wax"  or  "  Ijayberry  wax," 

^  Place  2  or  3  drops  in  a  test-tube  full  of  water.  If  saponification  is  complete  the  prod- 
ucts will  remain  in  solution  and  no  oil  will  separate. 

^  Under  some  conditions  a  purer  product  is  obtained  if  the  soap  solution  is  cooled  before 
precipitating  the  fatty  acid. 


FATS. 


145 


mflic  acid.  Remove  the  cake,  break  it  iato  small  pieces,  wash  it  with 
water  by  decantation  and  transfer  to  a  small  beaker  by  means  of  95  per 
cent  alcohol.  Heat  on  a  water-bath  until  the  palmitic  acid  is  dissolved, 
then  filter  through  a  dry  filter  paper  and  allow  the  filtrate  to  cool  slowly  in 
order  to  obtain  satisfactory  crystals.  Write  the  reactions  which  have 
taken  place  in  this  experiment. 

When  the  palmitic  acid  has  completely  crystallized  filter  off  the 
alcohol,  dry  the  crystals  between  the  filter  papers  and  try  the  tests 
given  in  E.xperiment  9,  below. 


Fig.  39 — Palmitic  Acid. 


8.  Salting-out  Experiments. — To  25  c.c.  of  soap  solution,  pre- 
pared as  described  above,  add  solid  sodium  chloride  to  the  point  of 
saturation,  with  continual  stirring.  A  menstruum  is  thus  formed  in 
which  the  soap  is  insoluble.  This  salting-out  process  is  entirely  anal- 
ogous to  the  salting-out  of  proteins  (see  page  106). 

9.  Palmitic  Acid. — (a)  Examine  the  crystals  under  the  microscope 
and  compare  them  with  those  shown  in  Fig.  39,  above. 

(b)  Solubility. — Try  the  solubility  of  palmitic  acid  in  the  same  sol- 
vents as  used  on  fats  (see  page  143). 

(c)  Melting-point. — Determine  the  melting-point  of  palmitic  acid 
by  one  of  the  methods  given  on  page  146. 

(d)  Formation  of  Transparent  Spot  on  Paper. — Melt  a  little  of  the  fatty 
acid  and  allow  a  drop  to  fall  upon  a  piece  of  ordinary  writing  paper. 
How  does  this  compare  with  the  action  of  a  fat  under  similar  circum- 
stances ? 

{e)  Acrolein  Test. — Apply  the  test  as  given  under  4,  page  143.  Explain 
the  result. 


146 


PHYSIOLOGICAL   CHEMISTRY. 


10.  Saponification  of  Lard. — To  25  grams  of  lard  in  a  flask  a'dd 
75  c.c.  of  alcoholic-potash  solution  and  warm  upon  a  water-bath  until 
saponification  is  complete.  (This  point  is  indicated  by  the  complete 
solubility  of  a  drop  of  the  solution  when  allowed  to  fall  into  a  little  water.) 
Now  transfer  the  solution  from  the  flask  to  an  evaporating  dish  con- 
taining about  100  c.c.  of  water  and  heat  on  a  water-bath  until  all  the 

alcohol  has  been  driven  off.  Precipitate 
the  fatty  acid  with  hydrochloric  acid  and 
cool  the  solution.  Remove  the  fatty  acid 
which  rises  to  the  surface,  neutralize  the 
solution  with  sodium  carbonate  and  evap- 
orate to  dryness.  Extract  the  residue  with 
alcohol,  remove  the  alcohol  by  evaporation 
upon  a  water-bath  and  on  the  residue  of 
glycerol  thus  obtained  make  the  tests  as 
given  below. 

II.  Glycerol,    (a)   Taste. — What  is  the 
taste  of  glycerol  ? 

(h)  Solubility. — Try    the    solubility    of 
glycerol  in  water,  alcohol  and  ether. 

(c)  Acrolein  Test. — Repeat  the  test  as 
given  under  4,  page  143. 

(rf)   Borax  Fusion    Test. — Fuse  a  little 
glycerol    on    a   platinum   wire   with    some 
powdered  borax  and  note  the  character 
istic  green  flame.     This  color  is  due  to  the 
glycerol  ester  of  boric  acid. 

(e)  Fehling's  Test. — How  does  this  re- 
sult compare  with  the  results  on  the  sugars  ? 
(/)  Solution  of  Cu (OH),. —Form  a  httle 
cupric  hydroxide  by  mixing  copper  sulphate 
and   potassium   hydroxide.      Add   a   httle 
glycerol  to  this  suspended  precipitate  and  note  what  occurs. 

12.  Melting-Point  of  fat.  First  Method.— Insert  one  of  the  melt- 
ing-point tubes,  furnished  by  the  instructor,  into  the  hquid  fat  and  draw 
up  the  fat  until  the  bulb  of  the  tube  is  about  one-half  full  of  the  material. 
Then  fuse  one  end  of  the  tube  in  the  flame  of  a  bunsen  burner  and  fas- 
ten the  tube  to  a  thermometer  by  means  of  a  rubber  band  in  such  a  manner 
that  the  bottom  of  the  fat  column  is  on  a  level  with  the  bulb  of  the  ther- 
mometer (Fig.  40,  above).  Fill  a  beaker  of  medium  size  about  two- 
thirds  full  of  water  and  place  it  within  a  second  larger  beaker  which 
also  contains  water,  the  two  vessels  being  separated  by  pieces  of  cork. 


Fig.  40. — Meltixg-Point 
Apparatus. 


FATS.  147 

Immerse  the  bulb  of  the  thermometer  and  the  attached  tube  in  such  a 
way  that  the  bulb  is  al^out  midway  between  the  upper  and  the  lower 
surfaces  of  the  water  of  the  inner  beaker.  The  upper  end  of  the  tube 
being  open  it  must  extend  above  the  surface  of  the  surrounding  water. 
Apply  gentle  heat,  stir  the  water,  and  note  the  temperature  at  which 
the  fat  first  begins  to  melt.  This  point  is  indicated  by  the  initial 
transparency.  For  ordinary  fats,  raise  the  temperature  very  cautiously 
from  30°  C.  To  determine  the  congealing-point  remove  the  flame  and 
note  the  temperature  at  which  the  fat  begins  to  solidify.  Record  the 
melting-  and  congealing-points  of  the  various  fats  submitted  by  the 
instructor. 

Second  Method. — Fill  a  small  evaporating  dish  about  one-half  full 
of  mercury  and  place  it  on  a  water-bath.  Put  a  small  drop  of  the  fat 
under  examination  on  an  ordinary  cover  glass  and  place  this  upon  the 
surface  of  the  mercury.  Raise  the  temperature  of  the  water-bath  slowly 
and  by  means  of  a  thermometer  whose  bulb  is  immersed  in  the  mercury, 
note  the  melting-point  of  the  fat.  Determine  the  congealing-point  by 
removing  the  flame  and  leaving  the  fat  drop  and  coverglass  in  position 
upon  the  mercury.  How  do  the  melting-points  as  determined  by  this 
method  compare  with  those  as  determined  by  the  first  method  ?  Which 
method  is  the  more  accurate,  and  why  ? 


CHAPTER  VIII. 
PANCREATIC  DIGESTION. 

As  soon  as  the  food  mixture  leaves  the  stomach  it  comes  into  inti- 
mate contact  with  the  bile  and  the  pancreatic  juice.  Since  these  fluids 
are  alkaline  in  reaction  there  can  obviously  be  no  further  peptic  activity- 
after  they  have  become  intimately  mixed  with  the  chyme  and  have 
neutralized  the  acidity  previously  imparted  to  it  by  the  hydrochloric 
acid  of  the  gastric  juice.  The  pancreatic  juice  reaches  the  intestine 
through  the  duct  of  Wirsung  which  opens  into  the  intestine  near  the 
pylorus. 

Normally  the  secretion  of  pancreatic  juice  is  brought  about  by  the 
stimulation  produced  by  the  acid  chyme  as  it  enters  the  duodenum. 
Therefore,  any  factor  which  produces  an  increased  flow  of  gastric  juice 
such,  for  example,  as  water ^  will  cause  a  stimulation  of  the  pancreatic 
secretion.  The  secretion  of  pancreatic  juice  is  probably  not  due  to  a  nerv- 
ous reflex  as  was  believed  by  Pawlow  but  rather,  as  Bayliss  and  Starling 
have  shown,  is  dependent  upon  the  presence,  in  the  epithelial  cells  of 
the  duodenum  and  jejunum  of  a  body  known  as  prosecretin.  This  body 
is  changed  into  secretin  through  the  hydrolytic  action  of  the  acid  present 
in  the  chyme.  The  secretin  is  then  absorbed  by  the  blood,  passes  to  the 
pancreas  and  stimulates  the  pancreatic  cells,  causing  a  flow  of  pancreatic 
juice.  The  quantity  of  juice  secreted  under  these  conditions  is  propor- 
tional to  the  amount  of  secretin  present.  The  activity  of  secretin  solutions 
is  not  diminished  by  boihng,  hence  the  body  does  not  react  like  an 
enzyme.  Further  study  of  the  body  may  show  it  to  be  a  definite  chem- 
ical individual  of  relatively  low  molecular  weight.  It  has  not  been 
possible  thus  far  to  obtain  secretin  from  any  tissues  except  the  mucous 
membrane  of  the  duodenum  and  jejunum. 

This  secretin  mentioned  above  belongs  to  the  class  of  substances 
called  hormones  or  chemical  messengers.  These  hormones  play  a  very 
important  part  in  the  coordination  of  the  activities  of  certain  functions 
and  glands.  Other  important  hormones  are  those  elaborated  by  the 
thyroids,  the  adrenals,  the  pituitary  body  (hypophysis),  the  embryo  and 
the  reproductive  glands.  It  is  claimed  that  all  active  organs  of  the  body 
produce  hormones. 

The  juice  as  obtained  from  a  permanent  fistula  differs  greatly  in 

'  .See  chapter  on  Gastric  Digestion. 

148 


PANCREATIC   DIGESTION.  1 49 

its  properties  from  the  juice  as  obtained  from  a  temporary  fistula,  and 
neither  form  of  fluid  possesses  the  properties  of  the  normal  fluid.  Pan- 
creatic juice  collected  by  Glaessner  from  a  natural  fistula  has  been  found 
to  be  a  colorless,  clear,  strongly  alkaline  fluid  which  foams  readily.  It  is 
further  characterized  by  containing  albumin,  globulin,  proteose,  and  pep- 
tone; nucleoprotein  is  also  present  in  traces.^  The  average  daily  secre- 
tion of  pancreatic  juice  is  650  c.c.  and  its  specific  gravity  is  1.008.  The 
fluid  contains  1.3  per  cent  of  solid  matter  and  the  freezing-point  is  — 0.47° 
C.  The  normal  pancreatic  secretion  contains  at  least  four  distinct 
enzymes.  They  arc  trypsin,  a  proteolytic  enzyme;  pancreatic  amylase 
(amylopsin),  an  amylolytic  enzyme;  pancreatic  lipase  (steapsin),  a  fat- 
splitting  enzyme;  and  pancreatic  rennin,  a  milk-coagulating  enzyme. 
Lactase,  the  lactose-splitting  enzyme,  is  also  present  at  certain  times. 

The  most  important  of  the  four  enzymes  of  the  pancreatic  juice  is 
the  proteolytic  enzyme  trypsin.  This  enzyme  resembles  pepsin  in  so 
far  as  each  has  the  power  of  breaking  down  protein  material,  but  the 
trypsin  has  much  greater  digestive  power  and  is  able  to  cause  a  more 
complete  decomposition  of  the  complex  protein  molecule.  In  the 
process  of  normal  digestion  the  protein  constituents  of  the  diet  are  for 
the  most  part  transformed  into  proteoses  (albumoses)  and  peptones 
before  coming  in  contact  with  the  enzyme  trypsin.  This  is  not  abso- 
lutely essential  however,  since  trypsin  possesses  digestive  activity  suffi- 
cient to  transform  unaltered  native  proteins  and  to  produce  from  their 
complex  molecules  comparatively  simple  fragments.  Among  the  prod- 
ucts of  tryptic  digestion  are  proteoses,  peptones,  peptides,  leucine,  tyrosine, 
aspartic  acid,  glutamic  acid,  alanine,  phenylalanine,  glycocoll,  cystine, 
serine,  valine,  proline,  oxyproline,  isoleucine,  arginine,  lysine,  histidine,  and 
tryptophane.  (The  crystalline  forms  of  many  of  these  products  are  repro- 
duced in  Chapter  IV.)  Trypsin  does  not  occur  preformed  in  the  gland, 
but  exists  there  as  a  zymogen  called  trypsinogen  which  bears  the  same 
relation  to  trypsin  that  pepsinogen  does  to  pepsin.  Trypsin  has  never 
been  obtained  in  a  pure  form  and  therefore  very  little  can  be  stated 
definitely  as  to  its  nature.  The  enzyme  is  the  most  active  in  alkaline 
solution  but  is  also  active  in  neutral  or  slightly  acid  solutions.  Trypsin 
is  destroyed  by  mineral  acids  and  may  also  be  destroyed  by  comparatively 
weak  alkali  (2  per  cent  sodium  carbonate)  if  left  in  contact  for  a  suflS- 
ciently  long  time.  Trypsinogen,  on  the  other  hand,  is  more  resistant  to 
the  action  of  alkalis.  In  pancreatic  digestion  the  protein  does  not  swell 
as  is  the  case  in  gastric  digestion,  but  becomes  more  or  less  "honey- 
combed" and  it  finally  disintegrates. 

The  presence  of  active  pepsin  in  the  contents  of  the  intestine  has  been 

*  Glaessner:  Zeitschri/t/ur  physiologische  Chemie,  40,  476,  1904. 


150  PHYSIOLOGICAL   CHEMISTRY.  : 

demonstrated  very  recently  by  Abderhalden  and  Meyer.  ^  It  may 
possibly  be  that  pepsin  may  play  a  part  in  the  profound  intestinal  pro- 
teolysis which  has  up  to  this  time  been  assigned  to  trypsin  and  erepsin 
(see  chapter  on  Gastric  Digestion). 

The  pancreatic  juice  which  is  collected  by  means  of  a  fistula  pos- 
sesses practically  no  power  to  digest  protein  matter.  A  body  called 
enter okinase  occurs  in  the  intestinal  juice  and  has  the  power  of  converting 
trypsinogen  into  trypsin.  This  process  is  known  as  the  "activation"  of 
trypsinogen  and  through  it  a  juice  which  is  incapable  of  digesting  protein 
may  be  made  active.  Enterokinase  is  not  always  present  in  the  intestinal 
juice  since  it  is  secreted  only  after  the  pancreatic  juice  reaches  the  intes- 
tine. It  resembles  the  enzymes  in  that  its  activity  is  destroyed  by  heat, 
but  differs  materially  from  this  class  of  bodies  in  that  a  certain  quantity 
is  capable  of  activating  only  a  definite  quantity  of  trypsinogen.  It  is, 
however,  generally  classified  as  an  enzyme.  Enterokinase  has  been 
detected  in  the  higher  animals,  and  a  kinase  possessing  similar  properties 
has  been  shown  to  be  present  in  bacteria,  fungi,  impure  fibrin,  lymph 
glands,  and  snake-venom.  Mendel  and  Rettger^  and  others  have  demon- 
strated that  activation  of  trypsinogen  into  trypsin  may  be  brought  about 
in  the  gland  as  well  as  in  the  intestine  of  the  living  organism.  The  manner 
of  the  activation  in  the  gland  and  the  nature  of  the  body  causing  it  are 
unknown  at  present.     Prym^   denies  that  such  an  activation  occurs. 

Delezenne  claims  that  trypsinogen  may  be  activated  by  soluble 
calctMrn  salts.  He  reports  experiments  which  indicate  that  proteolytic- 
ally  inactive  pancreatic  juice,  obtained  directly  from  the  duct,  when 
treated  with  salts  of  this  character,  assumes  the  property  of  digesting 
protein  material.  This  process  by  which  the  trypsinogen  is  activated 
through  the  instrumentality  of  calcium  salts  is  very  rapid  and  is  desig- 
nated by  Delezenne  as  an  "explosion."  The  recent  suggestion  of 
Mays  that  there  may  possibly  be  several  precursors  of  trypsin  one  of 
which  is  activated  by  enterokinase  and  the  others  by  other  agents,  is 
of  interest  in  this  connection. 

Pancreatic  amylase  (amylopsin),  the  second  of  the  pancreatic  en- 
zymes, is  an  amylolytic  enzyme  which  possesses  somewhat  greater  diges- 
tive power  than  the  salivary  amylase  (ptyalin)  of  the  saliva.  As  its 
name  implies,  its  activity  is  confined  to  the  starches,  and  the  products 
of  its  amylolytic  action  are  dtxtrins  and  sugars.  The  sugars  are  prin- 
cipally iso-maltose  and  maltose  and  these  by  the  further  action  of  an 
inverting  enzyme  are  partly  transformed  into  dextrose. 

.*  Abderhalden  and  Meyer:  Zeit.  physiol.  Chem.,  74,  67,  191 1. 
*  Mendel  and  Rettger:  American  Journal  of  Physiology,  7. 
'Prym:  Pfliiger's  Archiv.,  104  and  107. 


PANCREATIC   DIGESTION.  I5I 

It  is  possible  that  the  saliva  as  a  digestive  fluid  is  not  absolutely 
essential.  The  salivary  amylase  (ptyalin)  is  destroyed  by  the  hydro- 
chloric acid  of  the  gastric  juice  and  is  therefore  inactive  when  the  chyme 
reaches  the  intestine.  Should  undigested  starch  be  present  at  this  point 
however,  it  would  be  quickly  transformed  by  the  active  pancreatic  amy- 
lase. This  enzyme  is  not  present  in  the  pancreatic  juice  of  infants  during 
the  first  few  weeks  of  life,  thus  showing  very  clearly  that  a  starchy  diet 
is  not  normal  for  this  period. 

The  pronounced  influence  of  electrolytes  upon  the  action  of  pancreatic 
amylase  and  other  amylases  has  been  demonstrated  many  times.  ^  In 
this  connection  Bierry^  has  very  recently  shown  that  the  removal  of 
electrolytes  from  pancreatic  juice  by  dialysis  yields  a  juice  which  possesses 
no  power  to  split  starch.  He  further  claims  that  the  CI  or  Br  ion  is  ''abso- 
lutely essential  to  the  activity  of  animal  amylases."  It  is  generally  rec- 
ognized that  the  presence  of  the  CI  ion  facilitates  amylolytic  action.' 

It  has  been  claimed  that  pancreatic  amylase  has  a  slight  digestive 
action  upon  unboiled  starch. 

The  extent  to  which  amylase  is  present  in  the  feces  has  been  taken  as 
the  index  of  pancreatic  activity. 

The  third  enzyme  of  the  pancreatic  juice  is  called  pancreatic  lipase 
(steapsin)  and  is  a  fat-splitting  enzyme.  It  has  the  power  of  splitting 
the  neutral  fats  of  the  food  by  hydrolysis,  into  fatty  acid  and  glycerol.  A 
typical  reaction  would  be  as  follows: 

C3H,(0-C,,H3,CO)3  +  3H30-3(C,,H3,COOH)  +  C3H,(OH)3. 

Palmitin.  Palmitic  acid.  Glycerol. 

Recent  researches  make  it  probable  that  fats  undergo  saponifica- 
tion to  a  certain  extent  prior  to  their  absorption.  The  fatty  acids  formed, 
in  part  unite  with  the  alkalis  of  the  pancreatic  juice  and  intestinal  secre- 
tion to  form  soluble  soaps;  in  part  they  are  doubtless  absorbed  dissolved 
in  the  bile.  Some  obsen'ers  believe  that  the  fats  may  also  be  absorbed 
in  emulsion — a  condition  promoted  by  the  presence  of  the  soluble  soaps. 
After  absorption  the  fatty  acids  are  re-synthesized  to  form  neutral  fats 
with  glycerol. 

It  has  been  demonstrated  that  lipase  acts  best  in  dilution.*  This 
fact  is  of  importance  when  considered  in  connection  ^\-ith  the  fact  that  in- 
gested fat  is  better  utilized  in  the  human  organism  when  large  volumes 
of  water  (looo  c.c.)  are  taken  with  meals. ^ 

'  For  the  literature  see  Kendall  and  Sherman:  Jour.  Am.  ChemSuc,  32,  10S7,  1910. 
-  Bierr)-:  Biochem.  Zeit.,  40,  357,  1912. 

^Wohlgemuth:  Biochem  Zeit.,  9,  10,  1908;  and  Kendall  and  Sherman:  Jow.  Am.  Cliem. 
Sac.,  32,  10S7,  1910. 

*  Bradley:  Jour.  Biol.  Chem.,  8,  251,  1910. 

*  Mattill  and  Hawk:  Jour.  Am.  Chem.Soc,  ^2,^  1978,  1911. 


152  PHYSIOLOGICAL    CHEMISTRY. 

Pancreatic  lipase  is  very  unstable  and  is  easily  rendered  inert  by  the 
action  of  acid.  For  this  reason  it  is  not  possible  to  prepare  an  extract 
ha\dng  a  satisfactory  fat-splitting  power  from  a  pancreas  which  has 
been  removed  from  the  organism  for  a  sufficiently  long  time  to  have 
become  acid  in  reaction. 

The  fourth  enzyme  of  the  pancreatic  juice  is  called  pancreatic  rennin. 
It  is  a  milk-coagulating  enzyme  whose  action  is  very  similar  to  that 
of  the  enzyme  gastric  rennin  found  in  the  gastric  juice.  It  is  supposed 
to  show  its  greatest  activity  at  a  temperature  varying  from  60°  to  65°  C. 
The  enzymes  of  the  intestinal  juice  {succus  entericus)  are  of  great 
importance  to  the  animal  organism.  These  enzymes  include  erepsin, 
sucrase,  maltase,  lactase,  and  enterokinase.  According  to  Boldyreff  lipase 
is  also  present. 

Erepsin  is  a  proteolytic  enzyme  which  has  the  property  of  acting 
upon  the  proteoses,  peptones  and  peptides  which  are  formed  through  the 
action  of  trypsin  and  further  splitting  them  into  amino  acids.  Erepsin 
has  no  power  of  digesting  any  native  proteins  except  caseinogen,  histones, 
and  protamines.  It  possesses  its  greatest  activity  in  an  alkaline  solution 
although  it  is  slightly  active  in  acid  solution.  An  extract  of  the  intestinal 
erepsin  may  be  prepared  by  treating  the  finely  divided  intestine  of  a 
cat,  dog,  or  pig  with  toluol-  or  chloroform-water  and  permitting  the 
mixture  to  stand  with  occasional  shaking  for  24-72  hours.  ^  Enzymes 
similar  to  erepsin  occur  in  various  tissues  of  the  organism. 

In  cases  of  gastric  cancer  a  peptide-splitting  enzyme  is  present  in 
the  stomach  contents.  The  glycyl-tryptophane  test  is  used  for  its 
detection  (see  chapters  on  Enzymes  and  Gastric  Digestion). 

The  three  invertases  sucrase,  maltase,  and  lactase  are  also  important 
enzymes  of  the  intestinal  mucosa.  The  sucrase  acts  upon  sucrose 
and  inverts  it  with  the  formation  of  ifivert  sugar  (dextrose  and  laevulose). 
Some  investigators  claim  that  sucrase  is  also  present  in  saliva  and  gastric 
juice.  It  probably  does  not  exist  normally  in  either  of  these  digestive 
juices,  however,  and  if  found  owes  its  presence  to  the  excretory  processes 
of  certain  bacteria.  Sucrases  may  also  be  obtained  from  several  vegetable 
sources.  For  investigational  purposes  it  is  ordinarily  obtained  from 
yeast  (see  p.  13 j.  It  exhibits  its  greatest  activity  in  the  presence  of  a 
slight  acidity  but  if  the  acidity  be  increased  to  any  extent  the  reaction  is 
inhibited. 

Lactase  is   an   enzyme   which   inverts   lactose   with   the  consequent 

formation  of  dextrose  and  galactose.     Its  action  is  entirely  analogous, 

in  type,  to  that  of  sucrase.     It  has  apparently  been  proven  that  lactase 

occurs  in  the  intestinal  mucosa  of  the  young  of  all  animals  which  suckle 

*  See  page  15. 


PANCREATIC    DIGESTION.  1 53 

their  offspring.'  It  may  also  occur  in  the  intestinal  mucosa  of  certain 
adult  animals  if  such  animals  be  maintained  u])on  a  ration  containing 
more  or  less  lactose.  Fischer  and  Armstrong  have  demonstrated  the 
reversible  action"  of  lactase. 

For  discussions  of  maltase  and  cnterokinase  sec  pages  62  and  150 
respectively. 

PREPARATION  OF  AN  ARTIFICIAL  PANCREATIC  JUICE.^ 

After  removing  the  fat  from  the  pancreas  of  a  pig  or  sheep,  finely 
divide  the  organ  by  means  of  scissors  and  grind  it  in  a  mortar.  If 
convenient,  the  use  of  an  ordinary  meat  chopper  is  a  very  satisfactory 
means  of  preparing  the  pancreas. 

When  finely  divided  as  above  the  pancreas  should  be  placed  in  a 
500  c.c.  flask,  about  150  c.c.  of  30  per  cent  alcohol  added  and  the  flask 
and  contents  shaken  frequently  for  tv^^enty-four  hours.  (What  is  the 
reaction  of  this  alcoholic  extract  at  the  end  of  this  period,  and  why?) 
Strain  the  alcoholic  extract  through  cheese  cloth,  filter,  nearly  neutralize 
with  potassium  hydroxide  solution  and  then  exactly  neutralize  it  with 
0.5  per  cent  sodium  carbonate. 

Products  of  Tryptic  Digestion. 

Take  about  200  grams  of  lean  beef  which  has  been  freed  from  fat 
and  finely  ground  and  place  it  in  a  large-sized  beaker.  Introduce 
equal  volumes  of  the  pancreatic  extract  prepared  as  above  and  0.5 
per  cent  sodium  carbonate,  add  5  c.c.  of  an  alcoholic  solution  of  thymol 
to  prevent  putrefaction,  and  place  the  beaker  in  an  incubator  at  40°  C. 
Stir  the  contents  of  the  beaker  frequently  and  add  more  thymol  if  it 
becomes  necessary.  Allow  digestion  to  proceed  for  from  2  to  5  days 
and  then  separate  the  products  formed  as  follows:  Strain  off  the  undis- 
solved residue  through  cheese  cloth,  nearly  neutralize  the  solution  with 
dilute  hydrochloric  acid  and  then  exactly  neutralize  it  with  0.2  per 
cent  hydrochloric  acid.  A  precipitate  at  this  point  would  indicate 
alkali  metaprotein  (alkali  albuminate).  Filter  off  any  precipitate  and 
divide  the  filtrate  into  two  parts,  a  one-fourth  and  a  three-fourth  portion. 

Transfer  the  one-fourth  portion  to  an  evaporating  dish  and  make 
the  separation  of  proteoses  and  peptones  as  well  as  the  final  tests  upon 
these  bodies  according  to  the  directions  given  on  page  120. 

Place  about  5  c.c.  of  the  three-fourth  portion  in  a  test-tube  and 

*  Mendel  and  Mitchell:  American  Journal  of  Physiology,  20,  81,  1907. 
-  See  p.  8. 

'For  other  methods  of  preparation  see  Karl  Mays:  Zeitschrift  fUr  physiologische  Chemie, 
38,  428,  1903. 


154  PHYSIOLOGICAL   CHEMISTRY. 

add  about  i  c.c.  of  bromine  water.  A  violet  coloration  indicates  the 
presence  of  tryptophane  (see  page  82.  Also  see  glycyl-tryptophane 
reaction  in  chapter  on  Enzymes.)  Concentrate^  the  remainder  of  the 
three-fourth  portion  to  a  thin  syrup  and  make  the  separation  of  leucine 
and  tyrosine  according  to  the  directions  given  on  page  82. 

GENERAL  EXPERIMENTS  ON  PANCREATIC  DIGESTION. 

Experiments  on  Trypsin. 

.    I.  The    Most    Favorable    Reaction    for    Tryptic    Digestion. — 

Prepare  seven  tubes  as  follows: 

{a)  2-3  c.c.  of  neutral  pancreatic  extract  +  2-3  c.c.  of  water. 

{b)  2-3  c.c.  of  neutral  pancreatic  extract  +  2-3  c.c.  of  i  per  cent 
sodium  carbonate. 

(c)  2-3  c.c.  of  neutral  pancreatic  extract  +  2-3  c.c.  of  0.5  per  cent 
sodium  carbonate. 

{d)  2-3  c.c.  of  neutrfil  pancreatic  extract  +  2-3  c.c.  of  0.2  per  cent 
hydrochloric  acid. 

{e)  2-3  c.c.  of  neutral  pancreatic  extract  +  2-3  c.c.  of  0.2  per  cent 
combined  hydrochloric  acid. 

(/)  2-3  c.c.  of  neutral  pancreatic  extract  +  2-3  c.c.  of  0.4  per  cent 
boric  acid. 

is)  2-3  c.c.  of  neutral  pancreatic  extract  +  2-3  c.c.  of  0.4  per  cent 
acetic  acid. 

Add  a  small  piece  of  fibrin  to  the  contents  of  each  tube  and  keep 
them  at  40°  C.  noting  the  progress  of  digestion.  In  which  tube  do 
we  find  the  most  satisfactory  digestion,  and  why  ?  How  do  the  indi- 
cations of  the  digestion  of  fibrin  by  trypsin  differ  from  the  indications 
of  the  digestion  of  fibrin  by  pepsin  ? 

2.  The  Most  Favorable  Temperature. — (For  this  and  the  fol- 
lowing series  of  experiments  under  tryptic  digestion  use  the  neutral 
extract  plus  an  equal  volume  of  0.5  per  cent  sodium  carbonate.)  In 
each  of  four  tubes  place  5  c.c.  of  alkaline  pancreatic  extract.  Immerse 
one  tube  in  cold  water  from  the  faucet,  keep  a  second  at  room  tempera- 
ture and  place  a  third  in  the  incubator  or  water-bath  at  40°  C.  Boil  the 
contents  of  the  fourth  for  a  few  moments,  then  cool  and  also  keep  it  at 
40°  C.  Into  each  tube  introduce  a  small  piece  of  fibrin  and  note  the 
progress  of  digestion.  In  which  tube  does  the  most  rapid  digestion  occur  ? 
What  is  the  reason  ? 

3.  Influence  of  Metallic  Salts,  Etc. — ^Prepare  a  series  of  tubes  and 
into  each  tube  place  6  volumes  of  water,  3  volumes  of  alkaline  pancre- 

'  If  the  solution  is  alkaline  in  reaction,  while  it  is  being  concentrated,  the  amino  acids 
will  be  broken  down  and  ammonia  will  be  liberated. 


PANCREATIC   DIGESTION.  1 55 

atic  extract  and  i  volume  of  one  of  the  chemicals  listed  in  Experiment 
1 8  under  Salivary  Digestion,  page  66. 

Introduce  a  small  piece  of  fibrin  into  each  of  the  tubes  and  keep 
them  at  40°  C.  for  one-half  hour.  Shake  the  tubes  frequently.  In 
which  tubes  do  we  get  the  least  digestion  ? 

4.  Influence  of  Bile. — Prepare  live  tubes  as  follows: 

(a)  Five  c.c.  of  pancreatic  extract  +   1/2-1  c.c.  of  bile. 

(b)  Five  c.c.  of  pancreatic  extract  +    1-2  c.c.  of  bile. 

(c)  Five  c.c.  of  pancreatic  extract  +   2-3  c.c.  of  bile. 

(d)  Five  c.c.  of  pancreatic  extract  +   5  c.c.  of  bile.  ' 

(e)  Five  c.c.  of  pancreatic  extract. 

Introduce  into  each  tube  a  small  piece  of  fibrin  and  keep  them  at 
40°  C.  Shake  the  tubes  frequently  and  note  the  progress  of  digestion. 
Does  the  presence  of  bile  retard  tryptic  digestion?  How  do  these 
results  agree  with  those  obtained  under  gastric  digestion  ? 

Experiments  on  Pancreatic  Amylase. 

1.  The  Most  Favorable  Reaction. — Prepare  seven  tubes  as  follows: 

(a)  One  c.c.  of  neutral  pancreatic  extract  +  i  c.c.  of  starch  paste  + 
2  c.c.  of  water. 

(b)  One  c.c.  of  neutral  pancreatic  extract  +  i  c.c.  of  starch  paste  + 
2  c.c.  of  I  per  cent  sodium  carbonate. 

(c)  One  c.c.  of  neutral  pancreatic  extract  +  i  c.c.  of  starch  paste  + 
2  c.c.  of  0.5  per  cent  sodium  carbonate. 

(d)  One  c.c.  of  neutral  pancreatic  extract  +  i  c.c.  of  starch  paste  + 
2  c.c.  of  0.2  per  cent  hydrochloric  acid. 

(e)  One  c.c.  of  neutral  pancreatic  extract  +  i  c.c.  of  starch  paste  + 
2  c.c.  of  0.2  per  cent  combined  hydrochloric  acid. 

(/)  One  c.c.  oi  neutral  pancreatic  extract  +  i  c.c.  of  starch  paste  + 
2  c.c.  of  0.4  per  cent  boric  acid. 

(g)  One  c.c.  of  neutral  pancreatic  extract  +  i  c.c.  of  starch  paste  + 
2  c.c.  of  0.4  per  cent  acetic  acid. 

Shake  each  tube  thoroughly  and  place  them  in  the  incubator  or  water- 
bath  at  40°  C.  At  the  end  of  a  half-hour  divide  the  contents  of  each  tube 
into  two  parts  and  test  one  part  by  the  iodine  test  and  the  other  part  bv 
Fehling's  test.  Where  do  you  find  the  most  satisfactory  digestion  ? 
How  do  the  results  compare  with  those  obtained  from  the  similar  series 
under  Trypsin,  page  154? 

2.  The  Most  Favorable  Temperature. — (For  this  and  the  fol- 
lowing series  of  experiments  upon  pancreatic  amylase  use  the  neutral 
extract  plus  an  equal  volume  of  0.5  per  cent  sodium  carbonate.)     In 


156  PHYSIOLOGICAL   CHEMISTRY. 

each  of  four  tubes  place  2-3  c.c.  of  alkaline  pancreatic  extract.  Immerse 
one  tube  in  cold  water  from  the  faucet,  keep  a  second  at  room  tempera- 
ture, and  place  a  third  on  the  water-bath  at  40°  C.  Boil  the  contents 
of  the  fourth  for  a  few  moments,  then  cool  and  also  keep  it  at  40°  C. 
Into  each  tube  introduce  2-3  c.c.  of  starch  paste  and  note  the  progress 
of  digestion.  At  the  end  of  one-half  hour  divide  the  contents  of  each 
tube  into  two  parts  and  test  one  part  by  the  iodine  test  and  the  other 
part  bv  Fehling's  test.  In  which  tube  do  you  find  the  most  satisfactory 
digestion?  How  does  this  result  compare  with  the  result  obtained 
in  the  similar  series  of  experiments  under  Trypsin  (see  page  154)  ? 

3.  Influence  of  Metallic  Salts,  etc. — Prepare  a  series  of  tubes 
and  into  each  place  3  volumes  of  water,  3  volumes  of  alkaline  pancreatic 
extract,  i  volume  of  one  of  the  chemicals  listed  in  Experiment  18  under 
Salivary  Digestion,  page  66,  and  3  volumes  of  starch  paste.  Be  sure 
to  introduce  the  starch  paste  into  the  tube  last.  Why?  Shake  the 
tubes  well  and  place  them  in  the  incubator  or  water-bath  at  40°  C.  At  the 
end  of  a  half-hour  divide  the  contents  of  each  tube  into  two  parts  and  test 
one  part  by  the  iodine  test  and  the  other  part  by  Fehling's  test.  What 
are  your  conclusions  ? 

4.  Influence  of  Bile. — Prepare  five  tubes  as  follows: 

(a)  2-3  c.c.  of  pancreatic  extract  -|-  2-3  c.c.  of  starch  paste  -f  1/2-1 
c.c.  of  bile. 

(b)  2-3  c.c.  of  pancreatic  extract  -h  2-3  c.c.  of  starch  paste  -j-  1-2 
c.c.  of  bile. 

(c)  2-3  c.c.  of  pancreatic  extract  +  2-3  c.c.  of  starch  paste  4-  2-3 
c.c.  of  bile. 

(d)  2-3  c.c.  of  pancreatic  extract  -|-  2-3  c.c.  of  starch  paste  -f  5  c.c. 
of  bile. 

(e)  2-3  c.c.  of  pancreatic  extract  -|-  2-3  c.c.  of  starch  paste. 

Shake  the  tubes  thoroughly  and  place  them  in  the  incubator  or  water- 
bath  at  40°  C.  Note  the  progress  of  digestion  frequently  and  at  the  end 
of  a  half-hour  divide  the  contents  of  each  tube  into  two  parts  and  test  one 
part  by  the  iodine  test  and  the  other  part  by  Fehling's  test.  What  are 
your  conclusions  regarding  the  influence  of  bile  upon  the  action  of 
pancreatic  amylase  ? 

5.  Digestion  of  Dry  Starch. — To  a  little  dry  starch  in  a  test-tube 
add  about  5  c.c.  of  pancreatic  extract  and  place  the  tube  in  the  incubator 
or  water-bath  at  40°  C.  At  the  end  of  a  half-hour  filter  and  test  separate 
portions  of  the  filtrate  by  the  iodine  and  Fehling  tests.  What  do  you  con- 
clude regarding  the  action  of  pancreatic  amylase  upon  dry  starch? 
Compare  this  result  with  that  obtained  in  the  similar  experiment  under 
Salivary  Digestion  (page  65). 


PANCREATIC   DIGESTION.  1 57 

6.  Digestion  of  Inulin. — To  5  c.c.  of  inulin  solution  in  a  test-tube 
add  10  drops  of  pancreatic  extract  and  place  the  tube  in  the  incubator  or 
water-bath  at  40°  C.  After  one-half  hour  test  the  solution  by  Fehling's 
test/  Is  any  reducing  substance  present?  What  do  you  conclude 
regarding  the  digestion  of  inulin  by  pancreatic  amylase? 

Experiments  on  Pancreatic  Lipase. 

1.  "Litmus-milk"  Test. — Into  each  of  two  test-tubes  introduce 
10  c.c.  of  milk  and  a  small  amount  of  litmus  powder.  To^  the  con- 
tents of  one  tube  add  3  c.c.  of  neutral  pancreatic  extract  and  to  the  con- 
tents of  the  other  tube  add  3  c.c.  of  water  or  of  boiled  neutral  pancre- 
atic extract.  Keep  the  tubes  at  40°  C.  and  note  any  changes  which 
may  occur.     What  is  the  result  and  how  do  you  explain  it  ? 

2.  Ethyl  Butyrate  Test. — Into  each  of  two  test-tubes  introduce 
4  c.c.  of  water,  2  c.c.  of  ethyl  butyrate,  CgH^COO.CjH^,  and  a  small 
amount  of  litmus  powder.  To  the  contents  of  one  tube  add  4  c.c.  of 
neutral  pancreatic  extract  and  to  the  contents  of  the  other  tube  add  4 
c.c.  of  water  or  of  boiled  neutral  pancreatic  extract.  Keep  the  tubes  at 
40°  C.  and  observe  any  changes  which  may  occur.  What  is  the  result 
and  how  do  you  explain  it  ?  Write  the  equation  for  the  reaction  which 
has  taken  place. 

Experiments  on  Pancreatic  Rennin. 

Prepare  four  test-tubes  as  follows : 

(a)  Five  c.c.  of  milk  -t-  10  drops  of  neutral  pancreatic  extract. 

(b)  Five  c.c.  of  milk  -|-  20  drops  of  neutral  pancreatic  extract. 

(c)  Five  c.c.  of  milk  +  10  drops  of  a/)^a//;;f  pancreatic  extract. 
{d)  Five  c.c.  of  milk  +  20  drops  of  a/^a/m^ pancreatic  extract. 

Place  the  tubes  at  6o°-65°  C.  for  a  half  hour  ivithout  shaking.  Note 
the  formation  of  a  clot.^  How  does  the  action  of  pancreatic  rennin 
compare  with  the  action  of  the  gastric  rennin  ? 

'  If  the  inulin  solution  gives  a  reduction  before  being  acted  upon  by  the  pancreatic  juice, 
it  will  be  necessary  to  determine  the  extent  of  the  original  reduction  by  means  of  a  "check" 
test  (see  page  52). 

^  This  reaction  will  not  always  succeed,  owing  to  conditions  which  are  not  well 
understood. 


CHAPTER  IX. 
BILE. 

The  bile  is  secreted  continuously  by  the  liver  and  passes  into  the 
intestine  through  the  common  bile  duct  which  opens  near  the  pylorus. 
Bile  is  not  secreted  continuously  into  the  intestine.  In  a  fasting  animal 
no  bile  enters  the  intestine,  but  when  food  is  taken  the  bile  begins  to 
flow;  the  length  of  time  elapsing  between  the  ingestion  of  the  food  and 
the  secretion  of  the  bile  as  well  as  the  qualitative  and  quantitative  charac- 
teristics of  the  secretion  depending  upon  the  nature  of  the  food  ingested. 
Fats,  the  extractives  of  meat  and  the  protein  end-products  of  gastric 
digestion  (proteoses  and  peptones),  cause  a  copious  secretion  of  bile, 
whereas  such  substances  as  water,  acids  and  boiled  starch  paste  fail 
to  do  so.  In  general  a  rich  protein  diet  is  supposed  to  increase  the 
amount  of  bile  secreted,  whereas  a  carbohydrate  diet  would  cause  a 
much  less  decided  increase  and  might  even  tend  to  decrease  the  amount. 
It  has  been  demonstrated  by  Bayliss  and  Starling  that  the  secretion  of 
bile  is  under  the  control  of  the  same  mechanism  that  regulates  the  flow 
of  pancreatic  juice  (see  p.  148).  In  other  words,  the  hydrochloric 
acid  of  the  chyme,  as  it  enters  the  duodenum  transforms  prosecretin 
into  secretin  and  this  in  turn  enters  the  circulation,  is  carried  to  the 
liver,  and  stimulates  the  bile-forming  mechanism  to  increased  activity. 

We  may  look  upon  the  bile  as  an  excretion  as  well  as  a  secretion.  In 
the  fulfillment  of  its  excretory  function  it  passes  such  bodies  as  lecithin, 
metallic  substances,  cholesterol,  and  the  decomposition  products  of 
haemoglobin  into  the  intestine  and  in  this  way  aids  in  removing  them 
from  the  organism.  The  bile  assists  materially  in  the  absorption  of 
fats  from  the  intestine  by  its  solvent  action  on  the  fatty  acids  formed 
by  the  action  of  the  pancreatic  juice. 

The  bile  is  a  ropy,  viscid  substance  which  is  alkaline  in  reaction 
to  litmus,^  and  ordinarily  possesses  a  decidedly  bitter  taste.  It  varies 
in  color  in  the  different  animals,  the  principal  variations  being  yellow, 
brown,  and  green.  Fresh  human  bile  from  the  living  organism  ordi- 
narily has  a  green  or  golden-yellow  color.  Postmortem  bile  is  variable 
in  color.  It  is  very  difficult  to  determine  accurately  the  amount  of 
normal  bile  secreted  during  any  given  period.  For  an  adult  man  it 
has  been  variously  estimated  at  from  500  c.c.  to  iioo  c.c.  for  twenty- 

'  It  does  not  contain  any/^ee  hydroxyl  ions,  however. 

158 


BILE.  J  59 

four  hours.  The  specific  gravity  of  the  bile  varies  between  i.oio  and 
1.040,  and  the  freezing-point  is  about  — 0.56°  C.  As  secreted  by  the 
liver,  the  bile  is  a  clear,  limpid  fluid  which  contains  a  relatively  low 
content  of  solid  matter.  Such  bile  would  hav^e  a  specific  gravity  of 
approximately  i.oio.  After  it  reaches  the  gall-bladder,  however,  it 
becomes  mixed  with  mucous  material  from  the  walls  of  the  gall-bladder, 
and  this  process  coupled  with  the  continuous  absorption  of  water  from 
the  bile  has  a  "tendency  to  concentrate  the  secretion.  Therefore  the 
bile  as  we  find  it  in  the  gall-bladder,  ordinarily  possesses  a  higher  spe- 
cific gravity  than  that  of  the  freshly  secreted  fluid.  The  specific  grav- 
vity  under  these  conditions  may  run  as  high  a  1.040. 

The  principal  constituents  of  the  bile  are  the  salts  of  the  bile  acids, 
bile  pigments,  neutral  fats,  lecithin,  phosphatides,  and  cholesterol,  besides 
the  salts  of  iran,  copper,  calcium,  and  magnesium.  Zinc  has  also  fre- 
quently been  found  in  traces. 

The  bile  acids,  which  are  elaborated  exclusively  by  the  hepatic 
cells,  may  be  divided  into  two  groups,  the  glycocholic  acid  group  and 
the  taurocholic  acid  group.  In  human  bile  glycocholic  acid  predomi- 
nates, while  taurocholic  acid  is  the  more  abundant  in  the  bile  of  car- 
nivora.  The  bile  acids  are  conjugate  amino-acids,  the  glycocholic 
acid  yielding  glycocoll, 

CH^NH, 

COOH, 

and  cholic  acid  upon  decomposition,  whereas  taurocholic  acid  gives 
rise  to  taurine, 

CH,HN, 

CH.-SO^-OH, 

and  cholic  acid  under  like  conditions.  Glycocholic  acid  contains  some 
nitrogen  but  no  sulphur,  whereas  taurocholic  acid  contains  both  these 
elements.  The  sulphur  of  the  taurocholic  acid  is  present  in  the  taurine 
(amino-ethyl-sulphonic-acid),  of  which  it  is  a  characteristic  constituent. 
There  are  several  varieties  of  cholic  acid  and  therefore  we  have  several 
forms  of  glycocholic  and  taurocholic  acids,  the  variation  in  constitution 
depending  upon  the  nature  of  the  cholic  acid  which  enters  into  the  com- 
bination. The  bile  acids  are  present  in  the  bile  as  salts  of  one  of  the 
alkalis,  generally  sodium.  The  sodium  glycocholate  and  sodium  tau- 
rocholate  may  be  isolated  in  crystalline  form,  either  as  balls  or  rosettes 
of  fine  needles  or  in  the  form  of  prisms  having  ordinarily  four  or  six 
sides  (Fig.  41,  p.  160).     The  salts  of  the  bile  acids  are  dextro-rotatory. 


l6o  PHYSIOLOGICAL    CHEMISTRY. 

Among  other  properties  these  salts  have  the  power  of  holding  the  choles- 
terol and  lecithin  of  the  bile  in  solution. 

Hammarsten  has  demonstrated  a  third  group  of  bile  acids  in  the 
bile  of  the  shark.  This  same  group  very  probably  occurs  in  certain 
other  animals  also.  These  acids  are  very  rich  in  sulphur  and  resemble 
ethereal  sulphuric  acids  inasmuch  as  upon  treatment  with  boiling  hydro- 
chloric acid  they  yield  sulphuric  acid. 


Fig    41. — Bile  Salts. 

The  bile  pigments  are  important  and  interesting  biliary  constit- 
uents. The  following  have  been  isolated:  bilirubin,  biliverdin,  bili- 
fiiscin,  biliprasin,  bilihumin,  bilicyanin,  choleprasin,  and  choletelin.  Of 
these,  bilirubin  and  biliverdin  are  the  most  important  and  predominate  in 
normal  bile.  The  colors  possessed  by  the  various  varieties  of  normal 
bile  are  due  almost  entirely  to  these  two  pigments,  the  biliverdin  being 
the  predominant  pigment  in  greenish  bile  and  the  bilirubin  being  the 
principal  pigment  in  lighter  colored  bile.  The  pigments,  other  than 
the  two  just  mentioned,  have  been  found  almost  exclusively  in  biliary 
calculi  or  in  altered  bile  obtained  as  post-mortem  examinations. 

Bilirubin,  which  is  perhaps  the  most  important  of  the  bile  pigments, 
is  apparently  derived  from  the  blood  pigment,  the  iron  freed  in  the 
process  being  held  in  the  liver.  Bilirubin  has  the  same  percentage  com- 
position as  haematoporphyrin,  which  may  be  produced  from  haematin. 
It  is  a  specific  product  of  the  liver  cells,  but  may  also  be  formed  in  other 
parts  of  the  body.  The  pigment  may  be  isolated  in  the  form  of  a  reddish- 
yellow  powder  or  may  be  obtained  in  part,  in  the  form  of  reddish-yellow 
rhombic  plates  (Fig.  42,  p.  161)  upon  the  spontaneous  evaporation  of 
its  chloroform  solution.  The  crystalline  form  of  bilirubin  is  practically 
the  same  as  that  of  haematoidin.     It  is  easily  soluble  in  chloroform, 


BILE,  l6l 

somewhat  less  soluble  in  alcohol  and  only  slightly  soluble  in  ether  and 
benzene.  Bilirubin  has  the  power  of  combining  with  certain  metals, 
particularly  calcium,  to  form  combinations  which  are  no  longer  soluble 
in  the  solvents  of  the  unaltered  pigment.  Upon  long  standing  in  contact 
with  the  air,  the  reddish-yellow  bilirubin  is  oxidized  \nth  the  formation 
of  the  green  biliverdin.  Bilirubin  occurs  in  animal  fluids  as  soluble 
bilirubin-alkali._ 


I 


^  I 

Fig.  42. — Bilirubin    (H^vl^toidix).     (Ogden.) 

Solutions  of  bilirubin  exhibit  no  absorption-bands.  If  an  ammoniacal 
solution  of  bilirubin-alkali  in  water  is  treated  with  a  solution  of  zinc 
chloride,  however,  it  shows  bands  similar  to  those  of  bilicyanin  (Absorp- 
tion Spectra,  Plate  II),  the  two  bands  between  C  and  D  being  rather  well 
defined. 

Biliverdin  is  particularly  abundant  in  the  bile  of  herbivora.  It  is 
soluble  in  alcohol  and  glacial  acetic  acid  and  insoluble  in  water,  chloro- 
form, and  ether.  Biliverdin  is  formed  from  bihrubin  upon  oxidation.  It 
is  an  amorphous  substance,  and  in  this  differs  from  bilirubin  which  may 
be  at  least  partly  crystallized  under  proper  conditions.  Biliverdin  may 
be  obtained  in  the  form  of  a  green  powder.  In  common  with  bilirubin, 
it  may  be  converted  into  hydrobilirubin  by  nascent  hydrogen. 

The  neutral  solution  of  bilicyanin  or  cholecyanin  is  bluish-green  or 
steel-blue  and  possesses  a  blue  fluorescence,  the  alkaline  solution  is  green 
with  no  appreciable  flourescence  and  the  strongly  acid  solution  is  violet- 
blue.  The  alkaline  solution  exhibits  three  absorption-bands,  the  first 
a  dark,  well-defined  band  between  C  and  D,  somewhat  nearer  C;  the 
second  a  less  sharply-defined  band  extending  across  D  and  the  third  a 
rather  faint  band  between  E  and  F,  near  E  (Absorption  Spectra,  Plate  II). 
The  strongly  acid  solution  exhibits  two  absorption  bands,  both  lying  be- 
tween C  and  E  and  separated  by  a  narrow  space  near  D.  A  third  band, 
exceedingly  faint,  may  ordinarily  be  seen  between  b  and  F. 

Biliary  calculi,  otherwise  designated  as  biliary  concretions  or  gall 
stones,  are  frequently  formed  in  the  gall-bladder.     These  deposits  may 


1 62  PHYSIOLOGICAL    CHEMISTRY. 

be  divided  into  three  classes,  cholesterol  calculi,  pigment  calculi,  and 
calculi  made  up  almost  entirely  of  inorganic  material.  This  last  class 
of  calculus  is  formed  principally  of  the  carbonate  and  phosphate  of  calciurfi 
and  is  rarely  found  in  man  although  quite  common  to  cattle.  The  pigmii^iit 
calculus  is  also  found  in  cattle,  but  is  more  common  to  man  than  the 
inorganic  calculus.  This  pigment  calculus  ordinarily  consists  principally 
of  bilirubin  in  combination  with  calcium;  biliverdin  is  sometimes  found 
in  small  amount.  The  cholesterol  calculus  is  the  one  found  most  fre- 
quently in  man.  These  may  be  formed  almost  entirely  of  cholesterol, 
in  which  event  the  color  of  the  calculus  is  very  light,  or  they  may  contain 
more  or  less  pigment  and  inorganic  matter  mixed  with  the  cholesterol, 
which  tends  to  give  us  calculi  of  various  colors. 
For  discussion  of  cholesterol  see  page  270. 

Experiments  on  Bile. 

1.  Reaction. — Test  the  reaction  of  fresh  ox  bile  to  litmus,  phenol- 
phthalein  and  congo  red. 

2.  Nucleoprotein. — Acidify  a  small  amount  of  bile  with  dilute 
acetic  acid.  A  precipitate  of  nucleoprotein  forms.  Bile  acids  will  also 
precipitate  here  under  proper  conditions  of  acidity. 

3.  Inorganic  Constituents. — Test  for  chlorides,  sulphates,  and 
phosphates  (see  page  64). 

4.  Tests  for  Bile  Pigments,  {a)  Gmelin's  Test. — To  about  5  c.c. 
of  concentrated  nitric  acid  in  a  test-tube  add  2-3  c.c.  of  diluted  bile 
carefully  so  that  the  two  fluids  do  not  mix.  At  the  point  of  contact  note 
the  various  colored  rings,  green,  blue,  violet,  red  and  reddish-yellow. 
Repeat  this  test  with  different  dilutions  of  bile  and  observe  its  delicacy. 

{b)  Rosenbach^s  Modification  of  Gmelin's  Test. — Filter  5  c.c.  of  diluted 
bile  through  a  small  filter  paper.  Introduce  a  drop  of  concentrated 
nitric  acid  into  the  cone  of  the  paper  and  note  the  succession  of  colors  as 
given  in  Gmelin's  test. 

(c)  Nakayama's  Reaction. — -To  5  c.c.  of  diluted  bile  in  a  test-tube 
add  an  equal  volume  of  a  10  per  cent  solution  of  barium  chloride,  centrifu- 
gate  the  mixture,  pour  off  the  supernatant  fluid,  and  heat  the  precipitate 
with  2  c.c.  of  Nakayama's  reagent.^  In  the  presence  of  bile  pigments 
the  solution  assumes  a  blue  or  green  color. 

{d)  Huppert^s  Reaction. — Thoroughly  shake  equal  volumes  of  undiluted 
bile  and  milk  of  lime  in  a  test-tube.  The  pigments  unite  with  the  calcium 
and  are  precipitated.     Filter  off  the  precipitate,  wash  it  with  water,  and 

'  Prepared  by  combining  99  c.c.  of  alcohol  and  i  c.c.  of  fuming  hydrochloric  acid  con- 
taining 4  grams  of  ferric  chloride  per  liter. 


BILE.  163 

transfer  to  a  small  beaker.  Add  alcohol  acidified  slightly  with  hydro- 
chloric acid  and  warm  upon  a  water-bath  until  the  soluticm  becomes 
colored  an  emerald  green. 

In  examining  urine  for  bile  pigments,  according  to  Steensma,  this 
procedure  may  give  negative  results  even  in  the  presence  of  the  pigments, 
owing  to  the  fact  that  the  acid-alcohol  is  not  a  sufficiently  strong  oxidizing 
agent.  He  therefore  suggests  the  addition  of  a  drop  of  a  0.5  per  cent 
solution  of  sodium  nitrite  to  the  acid-alcohol  mixture  before  warming  on 
the  water-bath.     Try  this  modification  also. 

(e)  Hammarsten's  Reaction. — To  about  5  c.c.  of  Hammarsten's 
reagent^  in  a  small  evaporating  dish  add  a  few  drops  of  diluted  bile.  A 
green  color  is  produced.  If  more  of  the  reagent  is  now  added  the  play 
of  colors  as  observed  in  Gmelin's  test  may  be  obtained. 

(/)  Smith's  Test. — To  2-3  c.c.  of  diluted  bile  in  a  test-tube  add 
carefully  about  5  c.c.  of  dilute  tincture  of  iodine  (1:10)  so  that  the 
fluids  do  not  mix.  A  play  of  colors,  green,  blue  and  violet,  is  observed. 
In  making  this  test  upon  the  urine  ordinarily  only  the  green  color  is 
observed. 

(g)  Salkowski-S chipper s  Reaction. — To  10  c.c.  of  diluted  bile  in  a 
test-tube  add  5  drops  of  a  20  per  cent  solution  of  sodium  carbonate  and 
10  drops  of  a  20  per  cent  solution  of  calcium  chloride.  Filter  off  the  re- 
sultant precipitate  upon  a  hardened  filter-paper  and  wash  it  with  water. 
Remove  the  precipitate  to  a  small  porcelain  dish,  add  3  c.c.  of  an  acid- 
alcohol  mixture-  and  a  few  drops  of  a  dilute  solution  of  sodium  nitrite  and 
heat.  The  production  of  a  green  color  indicates  the  presence  of  bile 
pigments. 

Qi)  Bonanno's  Reaction.^ — Place  5-10  c.c.  of  diluted  bile  in  a  small 
porcelain  evaporating  dish  and  add  a  few  drops  of  Bonanno's  reagent.'* 
An  emerald-green  color  will  develop. 

5.  Tests  for  Bile  Acids,  (a)  Pettenkofer's  Test. — To  5  c.c.  of 
diluted  bile  in  a  test-tube  add  5  drops  of  a  5  per  cent  solution  of  sucrose. 
Now  run  about  2-3  c.c.  of  concentrated  sulphuric  acid  carefully  down 
the  side  of  the  tube  and  note  the  red  ring  at  the  point  of  contact.  Upon 
slightly  agitating  the  contents  of  the  tube  the  whole  solution  gradually 
assumes  a  reddish  color.  As  the  tube  becomes  warm,  it  should  be  cooled 
in  running  water  in  order  that  the  temperature  of  the  solution  may  not 
rise  above  70°  C. 

'  Hammarsten's  reagent  is  made  by  mixing  i  volume  of  25  per  cent  nitric  acid  and  19 
volumes  of  25  per  cent  hydrochloric  acid  and  then  adding  i  volume  of  this  acid  mixture 
to  4  volumes  of  95  per  cent  alcohol. 

^  Made  by  adding  5  c.c.  of  concentrated  hydrochloric  acid  to  95  c.c.  of  96  per  cent  alcohol. 

' //  Tommasi,  2,  No.  21. 

*  This  reagent  may  be  prepared  by  dissolving  2  grams  of  sodium  nitrite  in  100  c.c.  of 
concentrated  hydrochloric  acid. 


164  PHYSIOLOGICAL   CHEMISTRY. 

(b)  Mylius's  Modification  of.  Pettenkofer^s   Test. — To  approximately 

5  c.c.  of  diluted  bile  in  a  test-tube  add  3  drops  of  a  very  dilute  (1:1000) 

aqueous  solution  of  furfurol, 

HC  — CH 

II        II 
HC       CCHO. 


O 

Now  run  about  2-3  c.c.  of  concentrated  sulphuric  acid  carefully  down 
the  side  of  the  tube  and  note  the  red  ring  as  above.  In  this  case,  also, 
upon  shaking  the  tube  the  whole  solution  is  colored  red.  Keep  the 
temperature  of  the  solution  below  70°  C  as  before.. 

(c)  Neukomm's  Modification  of  Pettenkofer^s  Test. — To  a  few  drops 
of  diluted  bile  in  an  evaporating  dish  add  a  trace  of  a  dilute  sucrose 
solution  and  one  or  more  drops  of  dilute  sulphuric  acid.  Evaporate  on 
a  water-bath  and  note  the  development  of  a  violet  color  at  the  edge  of  the 
evaporating  mixture.  Discontinue  the  evaporation  as  soon  as  the  color 
is  observed. 

{d)  V.  Udrdnsky's  Test. — To  5  c.c.  of  diluted  bile  in  a  test-tube 
add  3-4  drops  of  a  very  dilute  (1:1,000)  aqueous  solution  of  furfurol. 
Place  the  thumb  over  the  top  of  the  tube  and  shake  the  tube  until  a  thick 
foam  is  formed.  By  means  of  a  small  pipette  add  2-3  drops  of  con- 
centrated sulphuric  acid  to  the  foam  and  note  the  dark  pink  coloration 
produced. 

(e)  Guerin's  Reaction. — To  equal  volumes  of  diluted  bile  and  alcohol 
in  a  test-tube  add  5-6  drops  of  a  saturated  aqueous  solution  of  furfurol 
and  5-6  drops  of  concentrated  sulphuric  acid.  A  blue  color  indicates 
bile  acids. 

if)  Hay's  Test. — This  test  is  based  upon  the  principle  that  bile  acids 
have  the  property  of  reducing  the  surface  tension  of  fluids  in  which  they 
are  contained.  The  test  is  performed  as  follows:  Cool  about  10  c.c.  of 
diluted  bile  in  a  test-tube  to  17°  C.  or  lower  and  sprinkle  a  Httle  finely 
pulverized  sulphur  upon  the  surface  of  the  fluid.  The  presence  of  bile 
acids  is  indicated  if  the  sulphur  sinks  to  the  bottom  of  the  liquid,  the 
rapidity  with  which  the  sulphur  sinks  depending  upon  the  quantity  of 
bile  acids  present  in  the  mixture.  The  test  is  said  to  react  with  bile 
acids  when  they  are  present  in  the  proportion  1:120,000. 

Some  investigators  claim  that  it  is  impossible  to  differentiate  between 
bile  acids  and  bile  pigments  by  this  test. 

6.  Crystallization  of  Bile  Salts. — To  25  c.c.  of  undiluted  bile  in 
an  evaporating-  dish  add  enough  animal  charcoal  to  form  a  paste  and 
evaporate  to  dryness  on  a  water-bath.     Remove  the  residue,  grind  it  in 


BILE. 


i6s 


a  mortar,  and  transfer  it  to  a  small  flask.  Add  about  50  c.c.  of  95  per 
cent  alcohol  and  boil  on  a  water-bath  for  20  minutes.  Filter,  and  add 
ether  to  the  filtrate  until  there  is  a  slight  permanent  cloudiness.  Cover 
the  vessel  and  stand  it  away  until  crystallization  is  complete.  Examine 
the  crystals  under  the  microscope  and  compare  them  with  those  shown 
in  Fig.  41,  page  160.  Try  one  of  the  tests  for  bile  acids  upon  some  of  the 
crystals. 

7.  Analysis  of  Biliary  Calculi. — Grind  the  calculus  in  a  mortar 
with  10  c.c.  of  ether.     Filter. 


Filtrate  I. 


Add  an  equal  volume  of  95  per  cent 
alcohol'  to  the  ether  extract,  allow  the 
mixture  to  evaporate  and  examine  for 
cholesterol  crj-stals  (Fig.  43,  p.  166). 
(For  further  tests  see  Experiment  8, 
below.)  


Residue  I. 
(On  paper  and  in  mortar.) 

Treat  with  dilute  hvdrochloric  acid  and 
filter. 


Filtrate  II.  Residue  II. 

Test  for  calcium,   phosphates,   and  (On  paper  and  in  mortar.) 

iron.     Evaporate  remainder  of  filtrate  Wash  with  a  little  water.     Dr\'  the  filter  paper. 
to  dryness  in  porcelain  crucible  and  I 

ignite.     Dissolve    residue     in     dilute  | 

hydrochloric  acid  and  make  alkaline  Treat  with  5  c.c.  chloroform  and  filter, 
with     ammonium     hydroxide.     Blue  I 

color  inilicates  copper.  . ■ ■ 

Filtrate  III.  Residue  III. 

Bilirubin.  (On  paper  and  in  mortar.) 

(Apply  test  for  j 

bile  pigments.)  | 

Treat    with    5    c.c.    of    hot 
alcohol  I 

Biliverdin. 

8.  Tests  for  Cholesterol. 

{a)  Microscopical  Examination. — Examine  the  crystals  under  the 
microscope  and  compare  them  with  those  shown  in  Fig.  43,  p.  166. 

(6)  Iodine-sulphuric  Acid  Test. — Place  a  few  crystals  of  cholesterol 
in  one  of  the  depressions  of  a  test-tablet  and  treat  with  a  drop  of  con- 
centrated sulphuric  acid  and  a  drop  of  a  very  dilute  solution  of  iodine. 
A  play  of  colors  consisting  of  violet,  blue,  green,  and  red  results. 

{c)  The  Liehermann-Burchard  Test. — Dissolve  a  few  crystals  of 
cholesterol  in  2  c.c.  of  chloroform  in  a  dry  test-tube.  Now  add  10 
drops  of  acetic  anhydride  and  1-3  drops  of  concentrated  sulphuric  acid. 
The  solution  becomes  red,  then  blue,  and  finally  bluish-green  in  color. 

'  The  alcohol  is  added  because  of  the  fact  that  it  is  often  found  that  crystallization  from 
pure  ether  does  not  yield  typical  cholesterol  crystals. 


i66 


PHYSIOLOGICAL   CHEMISTRY. 


(d)  Salkou'ski^s  Test. — Dissolve  a  few  crystals  of  cholesterol  in  a 
little  chloroform  and  add  an  equal  volume  of  concentrated  sulphuric 
acid.  A  play  of  colors  from  bluish-red  to  cherry-red  and  purple  is  noted 
in  the  chloroform  while  the  acid  assumes  a  marked  green  fluorescence. 

{e)  Schiff's  Reaction. — To  a  little  cholesterol  in  an  evaporating 
dish  add  a  few  drops  of  Schiff's  reagent.^  Evaporate  to  dryness  over 
a  low  flame  and  observe  the  reddish-violet  residue  which  changes  to  a 
bluish-violet. 


Fig.  43. — Cholesterol. 

9.  Preparation  of  Taurine. — To  300  c.c.  of  bile  in  a  casserole 
add  100  c.c.  of  hydrochloric  acid  and  heat  until  a  sticky  mass  (dyslysin) 
is  formed.  This  point  may  be  determined  by  drawing  out  a  thread- 
like portion  of  the  mass  by  means  of  a  glass  rod,  and  if  it  solidifies 
immediately  and  assumes  a  brittle  character  we  may  conclude  that  all 
the  taurocholic  and  glycocholic  acid  has  been  decomposed.  Decant 
the  solution  and  concentrate  it  to  a  small  volume  on  the  water-bath. 
Filter  the  hot  solution  to  remove  sodium  chloride  and  other  substances 
which  may  have  separated,  and  evaporate  the  filtrate  to  dryness.  Dis- 
solve the  residue  in  5  per  cent  hydrochloric  acid  and  precipitate  with 
ten  volumes  of  95  per  cent  alcohol.  Filter  off  the  taurine  and  recrystallize 
it  from  hot  water.  (Save  the  alcoholic  filtrate  for  the  preparation  of 
glycocoll,  below.)     Make  the  following  tests  upon  the  taurine  crystals. 

(a)  Examine  them  under  the  microscope  and  compare  with  Fig.  44. 

(b)  Heat  a  crystal  upon  platinum  foil.  The  taurine  at  first  melts, 
then  turns  brown,  and  finally  carbonizes  as  the  temperature  is  raised. 
Note  the  suffocating  odor.     What  is  it  ? 

*  Schiff's  reagent  consists  of  a  mixture  of  three  xolumes  of  concentrated  sulphuric  acid 
and  one  volume  of  lo  per  cent  ferric  chloride. 


BILK 


iby 


(c)  Test  the  solubility  of  the  crystals  in  water  and  in  alcohol. 

(d)  Grind  up  a  crystal  with  four  times  its  volume  of  dry  sodium 
carbonate  and  fuse  on  platinum  foil.  Cool  the  residue,  transfer  it  to 
a  test-tube,  and  dissolve  it  in  water.     Add  a  liltle  dilute  sulphuric    acid 


Fig.  44.. — Taurine. 


and  note  the  odor  of  hydrogen  sulphide.  Hold  a  piece  of  filter  paper, 
moistened  with  a  small  amount  of  lead  acetate,  over  the  opening  of 
the  test-tube  and  observe  the  formation  of  fead  sulphide. 


C 


/S?^^^'- 
<^^^>%^ 


Fig.  45. — Glycocoll. 


10.  Preparation  of  Glycocoll. — Concentrate  the  alcoholic  filtrate 
from  the  last  experiment  (9)  until  no  more  alcohol  remains.  The 
glycocoll  is  present  here  in  the  form  of  an  hydrochloride  and  may  be 
liberated  from  this  combination  by  the  addition  of  freshly  precipitated 


1 68  PHYSIOLOGICAL   CHEMISTRY. 

lead  hydroxide  or  by  lead  hydroxide  solution.  Remove  the  lead  by 
hydrogen  sulphide.  Filter  and  decolorize  the  filtrate  by  animal  charcoal. 
Filter  again,  concentrate  the  filtrate,  and  set  it  aside  for  crystallization. 
Glycocoll  separates  as  colorless  crystals  (Fig.  45.) 

II.  Synthesis  of  Hippuric  Acid. — To  some  of  the  glycocoll  pre- 
pared in  the  last  experiment  or  furnished  by  the  instructor,  add  a 
little  water,  about  i  c.c.  of  benzoyl  chloride  and  render  alkaline  with 
potassium  hydroxide  solution.  Stopper  the  tube  and  shake  it  until 
no  more  heat  is  evolved.  Now  render  strongly  alkaline  with  potassium 
hydroxide  and  shake  the  mixture  until  no  odor  of  benzoyl  chloride  can 
be  detected.  Cool,  acidify  with  hydrochloric  acid,  add  an  equal 
volume  of  petroleum  ether,  and  shake  thoroughly  to  remove  the  benzoic 
acid.  (Evaporate  this  solution  and  note  the  crystals  of  benzoic  acid. 
Compare  them  with  those  shown  in  Fig.  99,  page  308.)  Decant  the 
ethereal  solution  into  a  porcelain  dish  and  extract  again  with  ether. 
The  hippuric  acid  remains  in  the  aqueous  solution.  Filter  it  off  and 
wash  it  with  a  small  amount  of  cold  water  while  still  on  the  filter. 
Remove  it  to  a  small,  shallow  vessel,  dissolve  it  in  a  small  amount  of 
hot  water  and  set  it  aside  for  crystallization.  Examine  the  crystals 
microscopically  and  compare  them  with  those  in  Fig.  97,  page  300. 

The  chemistry  of  the  synthesis  is  represented  thus: 

CH,NH2  COCl     •        OC-NH-CH^-COOH. 

/\  /\ 

+  "^  i        I  +^^^- 

COOH  \/  \/ 

Glycocoll.  Benzoyl  chloride.        Hippuric  acta. 


CHAPTER  X. 
PUTREFACTION  PRODUCTS. 

The  putrefactive  processes  in  the  intestine  are  the  result  of  the 
action  of  bacteria  upon  the  protein  material  present.  This  bacterial 
action  which  is  the  combined  effort  of  many  forms  of  micro-organisms 
is  confined  almost  exclusively  to  the  large  intestine.  Some  of  the 
products  of  the  putrefaction  of  proteins  are  identical  vi^ith  those  formed 
in  tryptic  digestion,  although  the  decomposition  of  the  protein  material 
is  much  more  extensive  when  subjected  to  putrefaction.  Some  of  the 
more  important  of  the  putrefaction  products  are  the  following:  Indole, 
skatole,  paracresol,  phenol,  para-oxyphenylpropionic  acid,  para-oxyphenyl- 
acetic  acid,  volatile  fatty  acids,  hydrogen  sulphide,  methane,  methyl 
mercaptan,  hydrogen,  and  carbon  dioxide,  beside  proteoses,  peptones, 
ammonia,  and  amino  acids.  Of  these  the  indole,  skatole,  phenol,  and 
paracresol  appear  in  part  in  the  urine  as  ethereal  sulphuric  acids, 
whereas  the  oxyacids  mentioned  pass  unchanged  into  the  urine.  The 
potassium  indoxyl  sulphate  (page  298)  content  of  the  urine  is  a  rough 
indicator  of  the  extent  of  the  putrefaction  within  the  intestine. 

The  portion  of  the  indole  which  is  excreted  in  the  urine  is  first  sub- 
jected to  a  series  of  changes  within  the  organism  and  is  subsequently 
eliminated  as  indican.     These  changes  may  be  represented  thus : 


CH  /\  C(OH) 

+  0    -^ 
CH  \/\/CH 


NH  NH 

Indole-  Indoxyl- 


C(OH)  /\ CCO-SOgH) 

+  H3SO,   -^  +H,0 


CH  \/\/CH 

NH  NH 

Indoxyl.  Indoxyl  sulphuric  acid. 

In  the  presence  of  potassium  salts  the  indoxyl  sulphuric  acid  is  then 
transformed  into  indoxyl  potassium  sulphate  (or  indican), 

/\_    C(0S03K), 

\/\/CH 
NH 

and  eliminated  as  such  in  the  urine. 

169 


170  PHYSIOLOGICAL    CHEMISTRY. 

Indican  may  be  decomposed  by  treatment  with  concentrated  hydro- 
chloric acid  (see  tests  on  page  298)  into  sulphuric  acid  and  indoxyl. 
The  latter  body  may  then  be  oxidized  to  form  indigo-blue  thus: 

/\__C(OH)  /\ COOC     _/\ 

2  +2O--  4-2H20 

\  /  ^v/  CH  -xy  \/c=.  c\/\/ 

NH  NH  NH 

Indoxyl.  Indigo-blue. 

This  same  reaction  may  also  occur  under  pathological  conditions 
within  the  organism,  thus  giving  rise  to  the  appearance  of  crystals  of 
indigo-blue  in  the  urine. 

Skatole  is  hkewise  changed  within  the  organism  and  eliminated  in 
the  form  of  a  chromogenic  substance.  Skatole  is,  however,  of  less  impor- 
tance as  a  putrefaction  product  than  indole  and  ordinarily  occurs  in 
much  smaller  amount.  The  tryptophane  group  of  the  protein  molecule 
yields  the  indole  and  skatole  formed  in  intestinal  putrefaction,  but  the 
reasons  for  the  transformation  of  the  major  portion  of  this  tryptophane 
into  indole  and  the  minor  portion  into  skatole  are  not  well  understood. 
Indole  is  more  toxic  than  skatole. 

Phenol  occurs  in  fairly  large  amount  in  certain  abnormal  conditions 
of  the  organism,  but  ordinarily  the  amount  is  very  small.  It  is  probably 
derived  from  the  tyrosine  group  of  the  protein  molecule.  Phenol  is 
conjugated  in  the  liver  to  form  phenyl  potassium  sulphate  and  appears 
in  the  urine  in  this  form  (Baumann  and  Herter).  Para-cresol  occurs  in 
the  urine  as  cresyl  potassium  sulphate. 

Regarding  the  claim  of  Nencki  that  methyl  mercaptan  is  formed 
as  a  gas  during  intestinal  putrefaction  it  is  an  important  fact  that  Herter^ 
has  been  unable  to  detect  the  mercaptan  in  fresh  feces.  He  is,  therefore, 
not  inclined  to  accept  the  theory  that  methyl  mercaptan  is  formed  in 
ordinary  intestinal  putrefaction  but  beheves  that  it  may  be  formed  in 
exceptional  cases.  Hydrogen  sulphide  is,  however,  formed  in  all  cases  of 
intestinal  putrefaction. 

It  has  been  shown  by  Kutscher  and  his  associates^  that  many  acids 
and  bases  formed  in  putrefaction  and  which  have  been  considered  as 
originating  alone  from  bacterial  action,  may  also  be  formed  in  certain 
phases  of  metabolism  in  both  the  plant  and  animal  kingdom.  These 
transformation  products  of  amino  acids  have  been  termed  "aporrhegmas." 
The  following  aporrhegmas  may  result  from  putrefaction  processes: 

*  Herter:  Bacterial  Infections  of  the  Digestive  Tract,  p.  227. 

^  Ackermann  and  Kutscher;  Zeit.  physiol.  Ckem.,  69,  265,  1910. 

Ackermann:  Ibid.,  273. 

Engelanrl  anrl  Kutscher;  Ibid.,  282. 


PUTREFACTION    PRODUCTS.  I71 

Aporrhegma.  Amino  Acid  Source. 

Iminazolethylamine 1  Histidine. 

Iminazolpropionic  acid J 

Ornithine 1 

Tetramethylendiamine 1-  Arginine. 

Aminovalerianic  acid J 

Pentamethylendiamine Lysine. 

Amjnobutyric  acid Glutamic  acid. 

^'^"!"^-   • 1  Aspartic  acid. 

Succmic  acid J       ^ 

Isovalerianic  acid Leucine. 

Phenylethylamine 1 

Phenylaretic  acid [  Phenylalanine. 

Phenylpropionic  acid J 

/.-Oxyphenylacetic  acid. 1  Tyrosine. 

/'-O.xyphenylpropionic  acid J     ^ 

Indole 


Skatole.   .••■•:, \  Tryptophane. 

Indolacetic  acid -^^    ^ 

Indolpropionic  acid J 


Experiments  on  Putrefaction  Products. 

In  many  courses  in  physiological  chemistry  the  instructors  are  so 
limited  for  time  that  no  extended  study  of  the  products  of  putrefaction 
can  very  well  be  attempted.  Under  such  conditions  the  scheme  here 
submitted  may  be  used  profitably  in  the  way  of  demonstration.  Where 
the  number  of  students  is  not  too  great,  a  single  large  putrefaction  may 
be  started,  and,  after  the  initial  distillation,  both  the  resulting  distillate 
and  residue  may  be  distributed  to  the  members  of  the  class  for  individual 
manipulation. 

Preparation  of  Putrefaction  Mixture. — Place  a  weighed  mixture  of 
coagulated  egg  albumin  and  ground  lean  meat  in  a  flask  or  bottle  and 
add  approximately  2  liters  of  water  for  every  kilogram  of  protein  used. 
Sterilize  the  vessel  and  contents,  inoculate  with  the  colon  bacillus,  and 
keep  at  40°  C.  for  two  or  three  weeks.  If  cultures  of  the  colon  bacillus 
are  not  available,  add  60  c.c.  of  a  cold  saturated  solution  of  sodium 
carbonate  for  every  liter  of  water  previously  added  and  inoculate  with 
some  putrescent  material  (pancreas  or  feces). ^  Mix  the  putrefaction 
mixture  ver\'  thoroughly  by  shaking  and  insert  a  cork  furnished  with 
a  glass  tube  to  which  is  attached  a  wash  bottle  containing  a  3  per  cent 
solution  of  mercuric  cyanide.  ^  This  device  is  for  the  purpose  of  collecting 
the  methyl  mercaptan,  a  gas  formed  during  the  process  of  putrefaction. 
It  also  serves  to  diminish  the  odor  arising  from  the  putrefying  material. 
Place  the  putrefaction  mixture  at  40°  C.  for  two  or  three  weeks  and  at 

'  Putrefying  protein  may  be  prepared  by  treating  lo  grams  of  finely  ground  lean  meat 
with  100  c.c.  of  water  and  2  c.c.  of  a  saturated  solution  of  sodium  carbonate  and  keeping  the 
mixture  at  40°  C.  for  twenty-four  hours. 

-  Concentrated  sulphuric  acid  containing  a  small  amount  of  isatin  may  be  used  as  a 
substitute  for  mercuric  cyanide.  When  this  modification  is  employed  it  is  necessary  to  use 
calcium  chloride  tubes  to  e.xclude  moisture  from  the  isatin  solution. 


172  PHYSIOLOGICAL   CHEMISTRY. 

the  end  of  that  time  make  a  separation  of  the  products  of  putrefaction 
according  to  the  following  directions: 

Subject  the  mixture  to  distillation  until  the  distillate  and  residue  are 
approximately  equal  in  volume. 

PART  I. 
MANIPULATION  OF  THE  DISTILLATE. 

Acidify  with  hydrochloric  acid  and  extract  with  ether. 


Ether  Extract  No.  i.  Residue  No.  i. 

Add  an  equal  volume  of  water,  make  Allow  the  ether  to  volatilize.     Evapo 

alkaline  with  potassium  hydroxide,  and  rate     and     detect     ammonium     chloride 

shake  thoroughly.      I  crystals  (Fig.  46,  p.  173). 


Ether  Extract  No.  2.  Alkaline  Solution  No.  i. 

Evaporate  spontaneously.     Indole  and  Acidify    with    hydrochloric    acid,    add 

skatole    remain.     Try    proper    reactions  sodium     carbonate,     and     extract     with 

(see  pages  175  and  176).  ether. 


Ether  Extract  No.  3.  Alkaline  Solution  No.  2, 

Evaporate.     Detect  phenol  and  cresol  Acidify    with   hydrochloric    acid,    and 

(paracresol).     See  p.  177.  extract  with  ether. 


Ether  Extract  No.  4.  Final  Residue. 

Evaporate.     Volatile  fatty    acids    re-  (Discard.) 


DETAILED  DIRECTIONS  FOR  MAKING  THE  SEPARATIONS 
INDICATED  IN  THE  SCHEME. 

Preliminary  Ether  Extraction. — This  extraction  may  be  conveniently 
conducted  in  a  separatory  funnel.  Mix  the  fluids  for  extraction  in  the 
ratio  of  two  volumes  of  ether  to  three  volumes  of  the  distillate.  Shake 
very  thoroughly  for  a  few  moments,  then  draw  off  the  extracted  fluid 
and  add  a  new  portion  of  the  distillate.  Repeat  the  process  until  the 
entire  distillate  has  been  extracted.  Add  a  small  amount  of  fresh  ether 
at  each  extraction  to  replace  that  dissolved  by  the  water  in  the  preceding 
extraction. 

Residue  No.  1. — Unite  the  portions  of  the  distillate  extracted  as  above 
and  allow  the  ether  to  volatilize  spontaneously.  Evaporate  until  crystal- 
lization begins.  Examine  the  crystals  under  the  microscope.  Ammonium 
chloride  predominates.     Explain  its  presence. 

Ether  Extract  No.  i. — Add  an  equal  volume  of  water,  render  the 
mixture  alkaline  with  potassium  hydroxide,  and  shake  thoroughly  by 


PUTREFACTION   PRODUCTS. 


173 


means  of  a  separatory  funnel  as  before.  The  volatile  fatty  acids,  con- 
tained among  the  putrefaction  products,  would  be  dissolved  by  the  alka- 
line solution  (No.  i)  whereas  any  indole  or  skatole  would  remain  in  the 
ethereal  solution  (No.  2). 

Alkaline  Solution  No.  i. — Acidify  with  hydrochloric  acid  and  add 
sodium  carbonate  solution  until  the  fluid  is  neutral  or  slightly  acid 
from  the  presence  of  carbonic  acid.  At  this  point  a  portion  of  the 
solution,  after  being  heated  for  a  few  moments,  should  possess  an 
alkaline  reaction  on  cooling.  E.xtract  the  whole  mixture  with  ether 
in  the  usual  way,  using  care  in  the  manipulation  of  the  stop  cock  to 


Fig.  46. — .\mmoxium   Chloride. 

relieve  the  pressure  due  to  the  evolution  of  carbon  dioxide.  The  ether 
(Ether  Extract  No.  3)  removes  any  phenol  or  cresol  which  may  be  present 
while  the  volatile  fatty  acids  will  remain  in  the  alkaline  solution  (No.  2) 
as  alkali  salts. 

Ether  Extract  Xo.  2. — Drive  off  the  major  portion  of  the  ether  at  a 
low  temperature  on  a  water-bath  and  allow  the  residue  to  evaporate 
spontaneously.  Indole  and  skatole  should  be  present  here.  Prove  the 
presence  of  these  bodies.  For  tests  for  indole  and  skatole  see  pp.  175 
and  176. 

Alkaline  Solution  Xo.  2. — Make  strongly  acid  w^th  hydrochloric 
acid  and  extract  with  a  small  amount  of  ether,  using  a  separator}' 
funnel.  As  carbon  dioxide  is  liberated  here,  care  must  be  used  in  the 
manipulation  of  the  stop  cock  of  the  funnel  in  relie\-ing  the  pressure  within 
the  vessel.  The  volatile  fatty  acids  are  dissolved  by  the  ether  (Ether 
Extract  No.  4). 

Ether  Extract  Xo.  3. — Evaporate  this  ethereal  solution  on  a  water- 
bath.     The   oily  residue   contains  phenol   and   cresol.     The   cresol   is 


174  PHYSIOLOGICAL   CHEMISTRY. 

present  for  the  most  part  as  paracresol.  Add  some  water  to  the  oily 
residue  and  heat  it  in  a  flask.  Cool  and  prove  the  presence  of  phenol 
and  cresoL     For  tests  for  these  bodies  see  page  177. 

Ether  Extract  No.   4. — Evaporate  on  a   water-bath.     The   volatile 
fatty  acids  remain  in  the  residue. 


PART  II. 
MANIPULATION  OF  THE  RESIDUE. 

Evaporate,  filter,  and  extract  with  ether. 


Ether  Extract.  Aqueous  Solution. 

Evaporate,  extract  the  residue  with  Evaporate    until    crystals    begin    to 

warm  water,  and  filter.  form.     Stand    in    a    cold    place    until 

crystallization  is  complete.     Filter, 


Crystalline  Deposit.  Filtrate  No.  i. 

Consists    of    a    mixture    of  Contains     proteose^     peptone, 

leucine  and  tyrosine  crystals  aromatic  acids,  and  tryptophane. 

(Figs.  24,  27  and  109,  pages 
81,  85  and  367.) 


Filtrate  No.  2.  Residue. 

Contains    oxyacids    and  Contains  non-volatile 

skatole-carbonic  acid.  fatty  acids. 

DETAILED  DIRECTIONS  FOR  MAKING  THE 

SEPARATIONS  INDICATED  IN 

THE  SCHEME. 

Preliminary  Ether  Extraction. — This  extraction  may  be  conducted 
in  a  separatory  funnel.  In  order  to  make  a  satisfactory  extraction 
the  mixture  should  be  shaken  very  thoroughly.  Separate  the  ethereal 
solution  from  the  ac{ueous  portion  and  treat  them  according  to  the 
directions  given  on  p.  172. 

Ether  Extract. — Evaporate  this  solution  on  a  safety  water-bath  until 
the  ether  has  been  entirely  removed.  Extract  the  residue  with  warm 
water  and  filter. 

Aqueous  Solution. — Evaporate  this  solution  until  crystallization 
begins.  Stand  the  solution  in  a  cold  place  until  no  more  crystals  form. 
This  crystalline  mass  consists  of  impure  leucine  and  tyrosine.  Filter 
off  the  crystals. 

Crystalline  Deposit. — Examine  the  crystals  under  the  microscope 
and  compare  them  with  those  reproduced  in  Figs.  24,  27,  and  109,  pages 


PUTREFACTION    PRODUCTS.  1 75 

8i,  85  and  367.  Do  the  forms  of  the  crystals  of  leucine  and  tyrosine 
resemble  those  previously  examined  ?  Make  a  separation  of  the  leucine 
and  tyrosine  and  apply  typical  tests  according  to  directions  given  on 
pages  90  and  91. 

Filtrate  No.  i. — Make  a  test  for  tryptophane  with  bromine  water 
(see  page  153),  and  also  with  the  Hopkins-Cole  reagent  (see  page  98), 
Use  the  remainder  of  the  liltratc  for  the  separation  of  proteoses  and  pep- 
tones. Make  The  separation  according  to  the  directions  given  on 
page  120. 

Filtrate  No.  2. — This  solution  contains  para-oxyphenylacetic  acid, 
para-oxyphenylpropionic  acid  and  skatole-carbonic  acid.  Prove  the 
presence  of  these  bodies  by  appropriate  tests.  Tests  for  oxyacids  and 
skatole-carbonic  acid  are  given  on  page  177. 

TESTS  FOR  VARIOUS  PUTREFACTION  PRODUCTS. 
Tests  for  Indole. 

I.  Herter's  ;?-Naphthaquinone  Reaction. — (a)  To  a  dilute  aque- 
ous solution  of  indole  (1:500,000)  add  one  drop  of  a  2  per  cent  solution 
of  /3-naphthaquinone-sodium-monosulphonate.  No  reaction  occurs. 
Add  a  drop  of  a  10  per  cent  solution  of  potassium  hydroxide  and  note 
the  gradual  development  of  a  blue  or  blue-green  color  which  fades  to 
green  if  an  excess  of  the  alkali  is  added.  Render  the  green  or  blue-green 
solution  acid  and  note  the  appearance  of  a  pink  color.  Heat  fa- 
cilitates the  development  of  the  color  reaction. 

One  part  of  indole  in  one  million  parts  of  water  may  be  detected  by 
means  of  this  test  if  carefully  performed. 

{h)  If  the  alkali  be  added  to  a  more  concentrated  indole  solution 
before  the  introduction  of  the  naphthaquinone  the  course  of  the  re- 
action is  different,  particularly  if  the  indole  solution  is  somewhat  more 
concentrated  than  that  mentioned  above  and  if  heat  is  used.  Under 
these  conditions  the  blue  indole  compound  ultimately  forms  as  fine 
acicular  crystals  which  rise  to  the  surface. 

If  we  do  not  wait  for  the  production  of  the  crystalline  body  but  as 
soon  as  the  blue  color  forms,  shake  the  aqueous  solution  with  chloro- 
form, the  blue  color  disappears  from  the  solution  and  the  chloroform 
assumes  a  pinkish-red  hue.  This  is  a  distinguishing  feature  of  the  indole 
reaction  and  facilitates  the  differentiation  of  indole  from  other  bodies 
which  yield  a  similar  blue  color.  A  very  satisfactory  method  for  the 
quantitative  determination  of  indole  is  based  upon  the  principle  under- 
lying this  test. 


176  PHYSIOLOGICAL   CHEMISTRY. 

2.  Konto's  Reaction. — Distil  the  solution  to  be  tested  until  only 
one-third  of  the  original  solution  remains.  Make  the  distillate  alkaline 
with  sodium  hydroxide  and  distil  again  in  order  to  separate  the  indole  from 
the  phenol,  the  latter  remaining  in  the  residue.  Inasmuch  as  this  second 
distillate  generally  contains  a  large  amount  of  ammonia  it  should  be 
acidified  with  dilute  sulphuric  acid  and  again  distilled.  To  i  c.c.  of  this 
ammonia-free  distillate  in  a  test-tube  add  3  drops  of  a  40  per  cent  solution 
of  formaldehyde  and  i  c.c.  of  concentrated  sulphuric  acid.  Now  agitate 
the  mixture  and  note  the  appearance  of  a  violet  red  color  if  a  trace 
of  indole  is  present.  The  test  is  said  to  serve  for  the  detection  of  indole 
when  present  in  a  dilution  of  i :  700,000. 

Skatole  gives  a  yellow  or  brown  color  under  the  above  conditions. 

3.  Cholera-red  Reaction. — To  a  little  of  the  residue  in  a  test- 
tube  add  one-tenth  its  volume  of  a  0.02  per  cent  solution  of  potassium 
nitrite  and  mix  thoroughly.  Carefully  run  concentrated  sulphuric 
acid  down  the  side  of  the  tube  so  that  it  forms  a  layer  at  the  bottom. 
Note  the  purple  color.  Neutralize  with  potassium  hydroxide  and 
observe  the  production  of  a  bluish-green  color. 

\^i  4.  Legal's  Reaction. — -To  a  small  amount  of  the  residue  in  a  test- 
tube  add  a  few  drops  of  a  freshly  prepared  solution  of  sodium  nitro- 
prusside,  Na2Fe(CN)5NO-f2H20.  Render  alkaline  with  potassium 
hydroxide  and  note  the  production  of  a  violet  color.  If  the  solution  is 
now  acidified  with  glacial  acetic  acid  the  violet  is  transformed  into  a 
blue. 

5.  Pine  "Wood  Test. — Moisten  a  pine  splinter  with  concentrated 
hydrochloric  acid  and  insert  it  into  the  residue.  The  wood  assumes 
a  cherry-red  color. 

6.  Nitroso-indole  Nitrate  Test. — Acidify  some  of  the  residue 
with  nitric  acid,  add  a  few  drops  of  a  potassium  nitrite  solution  and 
note  the  production  of  a  red  precipitate  of  nitroso-indole  nitrate.  If 
the  residue  contains  but  little  indole  simply  a  red  coloration  will  result. 
Compare  this  result  with  the  result  of  the  similar  test  on  skatole. 

Tests  for  Skatole. 

I.  Herter's  Para-dimethylaminobenzaldehyde  Reaction.^ — To 
5  c.c.  of  the  distillate  or  aqueous  solution  under  examination  add 
I  c.c.  of  an  acid  solution  of  para-dimethylaminobenzaldehyde^  and 
heat  the  mixture  to  boiling.     A  purplish-blue  coloration  is  produced^ 

'  Herter:  Bacterial  hifeclions  of  the  Digestive  Tract,  1907,  p.  141. 

^  Made  by  dissolving   5   grams  of  para-dimethylaminobenzaldehyde  in    100  c.c.  of   10 
per  cent  sulphuric  acid. 

*  If  the  color  does  not  appear  add  more  of  the  aldehyde  solution. 


PUTREFACTION   PRODUCTS.  177 

which  may  be  intensified  through  the  addition  of  a  few  drops  of  concen- 
trated hydrochloric  acid.  If  the  solution  be  cooled  under  running  water 
it  loses  its  purplish  tinge  of  color  and  becomes  a  definite  blue.  The 
solution  at  this  point  may  be  somewhat  opalescent  through  the  separation 
of  uncombined  para-dimethlaminobenzaldehyde.  Care  should  be  taken 
not  to  add  an  excess  of  hydrochloric  acid  inasmuch  as  the  end-reaction 
has  a  tendency  fo  fade  under  the  influence  of  a  high  acidity. 

A  rough  idea  regarding  the  actual  quantity  of  skatole  in  a  mixture 
may  be  obtained  by  extracting  this  blue  solution  with  chloroform  and 
subsequently  comparing  this  chloroform  solution,  by  means  of  a  color- 
imeter (Duboscq),  with  the  maximal  reaction,  obtained  with  a  skatole 
solution  of  known  strength. 

2.  Color  Reaction  with  Hydrochloric  Acid. — Acidify  some  of 
the  residue  with  concentrated  hydrochloric  acid.  Note  the  production 
of  a  \iolet  color. 

3.  Acidify  some  of  the  residue  with  nitric  acid  and  add  a  few  drops 
of  a  potassium  nitrite  solution;  Note  the  white  turbidity.  Compare 
this  result  with  the  result  of  the  similar  test  on  indole. 

Tests  for  Phenol  and  Cresol. 

1.  Color  Test. — Test  a  little  of  the  solution  Avith  Millon's  reagent. 
A  red  color  results.  Compare  this  test  with  the  similar  one  under  Tyro- 
sine (see  page  91). 

2.  Ferric  Chloride  Test. — Add  a  few  drops  of  neutral  ferric  chloride 
solution  to  a  little  of  the  residual  fluid.    A  dirty  bluish-gray  color  is  formed. 

3.  Formation  of  Bromine  Compounds, — Add  some  bromine  water 
to  a  little  of  the  fluid  under  examination.  Note  the  crystalline  precipi- 
tate of  tribromphenol  and  tribromcresol. 

Tests  for  Oxyacids. 

1.  Color  Test. — Test  a  little  of  the  solution  with  Millon's  reagent. 
A  red  color  results. 

2.  Bromine  Water  Test. — Add  a  few  drops  of  bromine  water  to 
some  of  the  filtrate.     A  turbidity  or  precipitate  is  observed. 

Test  for  Skatole-carbonic  Acid. 

Ferric  Chloride  Test. — Acidify  some  of  the  filtrate  with  hydro- 
chloric acid,  add  a  few  drops  of  ferric  chloride  solution,  and  heat.  Com- 
pare the  end-reaction  with  that  given  by  phenol. 


CHAPTER  XI. 

FECES. 

The  feces  is  the  residual  mass  of  material  remaining  in  the  intes- 
tine after  the  full  and  complete  exercise  of  the  digestive  and  absorptive 
functions  and  is  ultimately  expelled  from  the  body  through  the  rectum. 
The  amount  of  this  fecal  discharge  varies  with  the  individual  and  the 
diet.  Upon  an  ordinary  mixed  diet  various  authorities  claim  that  the 
daily  excretion  by  an  adult  male  will  aggregate  1 10-170  grams  with  a 
solid  content  ranging  between  25  and  45  grams;  the  fecal  discharge  of 
such  an  individual  upon  a  vegetable  diet  will  be  much  greater  and  may 


Fig.  47. — Microscopical  Constituents  of  Feces,     (v.  Jaksch.) 
a,  Muscle  fibers;  b,  connective  tissue;  c,  epithelium;  d,  leucocytes;  e,  spiral  cells;/,  g,  h,  i, 
various  vegetable  cells;  k,  "triple  phosphate"  crystals;  /,  woody  vegetable  cells;  the  whole 
interspersed  with  innumerable  micro-organisms  of  various  kinds. 

even  be  as  great  as  350  grams  and  possess  a  solid  content  of  75  grams. 
In  the  author's  own  experience  the  average  daily  output  of  moist  feces, 
calculated  on  the  basis  of  data  secured  from  the  examination  of  over  1,000 
stools,  was  about  100  grams.  The  variation  in  the  normal  daily  output 
being  so  great  renders  this  factor  of  very  little  value  for  diagnostic  pur- 
poses, except  where  the  composition  of  the  diet  is  accurately  known. 
Lesions  of  the  digestive  tract,  a  defective  absorptive  function,  or  increased 
peristalsis  as  well  as  an  admixture  of  mucus,  pus,  blood,  and  pathological 
products  of  the  intestinal  wall  may  cause  the  total  amount  of  excrement 
to  be  markedly  increased. 

The   fecal   pigment   of  the   normal   adult  is  hydrobilirubin.     This 

178 


FECES.  179 

pigment  originates  from  the  bilirubin  which  is  secreted  into  the  intes- 
tine in  the  bile,  the  transformation  from  bilirubin  to  hydrobilirubin 
being  brought  about  through  the  activity  of  certain  bacteria.  Hydro- 
bilirubin is  sometimes  called  stercobilin  and  bears  a  close  resemblance 
to  urobilin  or  may  even  be  identical  with  that  pigment.  Neither  bilirubin 
nor  biliverdin  occurs  normally  in  the  fecal  discharge  of  adults,  although 
the  former  may  be  detected  in  the  excrement  of  nursing  infants.  The 
most  important  factor,  however,  in  determining  the  color  of  the  fecal 
discharge  is  the  diet.  A  mixed  diet,  for  instance,  produces  stools  which 
vary  in  color  from  light  to  dark  brown,  an  exclusive  meat  diet  gives 
rise  to  a  brownish-black  stool,  whereas 
the  stool  resulting  from  a  milk  diet  is 
invariably  light  colored.  Certain  pig- 
mented foods  such  as  the  chlorophyllic 
vegetables,  and  various  varieties  of  ber- 
ries, each  afford  stools  having  a  charac-  ^^ 

teristic  color.     Certain  drugs  act  in  a        !      ^^^  1  .^"*'*'    ^^J^\  / 
similar  way  to  color  the  fecal  discharge.  v^    J         «"  \-^   ' 

This  is  well  illustrated  bv  the  occurrence  "^        _  ^'^ 

.     I1G.48. — H^MATOiDLv  Crystals  FROM 
of    green    stools    following    the    use    ot  Acholic  Stools,    {v.  Jaksch.) 

calomel  and  of  black  stools  after  bismuth       Color  of  crystals  same  as  the  color  of 

those  in  Fig.  42,  p.  161. 

ingestion.       The    green    color    of    the 

calomel  stool  is  generally  believed  to  be  due  to  biliverdin.  v.  Jaksch, 
however,  claims  to  have  proven  this  view  to  be  incorrect  since  he  was 
able  to  detect  hydrobilirubin  (or  urobilin)  but  no  biliverdin  in  stools  after 
the  administration  of  calomel.  The  bismuth  stool  derives  its  color  from 
the  black  sulphide  which  is  formed  from  the  subnitrate  of  bismuth. 
In  cases  of  biliary  obstruction  the  grayish-white  acholic  stool  is  formed. 

Under  normal  conditions  the  odor  of  feces  is  due  to  skatole  and 
indole,  two  bodies  formed  in  the  course  of  putrefactive  processes  occurring 
within  the  intestine  (see  page  169).  Such  bodies  as  methane,  methyl 
mercaptan,  and  hydrogen  sulphide  may  also  add  to  the  disagreeable 
character  of  "the  odor.  The  intensity  of  the  odor  depends  to  a  large 
degree  upon  the  character  of  the  diet,  being  very  marked  in  stools  from 
a  meat  diet,  much  less  marked  in  stools  from  a  vegetable  diet,  and  fre- 
quently hardly  detectable  in  stools  from  a  milk  diet.  Thus  the  stool 
of  the  infant  is  ordinarily  nearly  odorless  and  any  decided  odor  may 
generally  be  readily  traced  to  some  pathological  source. 

A  neutral  reaction  ordinarily  predominates  in  normal  stools  although 
sHghtly  alkaline  or  even  acid  stools  are  met  with.  The  acid  reaction  is 
encountered  much  less  frequently  than  the  alkaline  and  then  commonly 
only  following  a  vegetable  diet. 


l8o  PHYSIOLOGICAL   CHEMISTRY. 

Recent  experiments^  in  which  the  actual  hydrogen  ion  concentration 
of  the  feces  was  determined  indicated  that  the  reaction  of  the  excreta 
was  uniformly  slightly  alkaline.  Pronounced  dietary  changes  e.  g.,  low 
protein  diet,  high  protein  diet,  fasting,  water  drinking  with  meals,  produced 
at  most  only  minor  changes  in  the  reaction  of  the  feces. 

The  form  and  consistency  of  the  stool  is  dependent,  in  large  measure, 
upon  the  nature  of  the  diet  and  particularly  upon  the  quantity  of  water 
ingested.  Under  normal  conditions  the  consistency  may  vary  from  a 
thin,  pasty  discharge  to  a  firmly  formed  stool.  Stools  which  are  ex- 
ceedingly thin  and  watery  ordinarily  have  a  pathological  significance. 
In  general  the  feces  of  the  carnivorous  animals  is  of  a  firmer  consistency 
than  that  of  the  herbivora. 

The  continued  ingestion  of  a  diet  which  is  very  thoroughly  digested 
and  absorbed  is  frequently  accompanied  by  the  formation  of  dry,  hard 
fecal  masses  (scybala).  Constipation  generally  results,  due  to  the  small 
bulk  of  the  feces  and  its  lack  of  moisture.  To  counteract  this  tendency 
toward  constipation  the  ingestion  of  agar-agar^  has  been  suggested.^  This 
agar  is  relatively  indigestible  and  readily  absorbs  water  thus  forming  a 
bulky  fecal  mass  which  is  sufficiently  soft  to  permit  of  easy  evacuation. 
The  function  of  agar  is  not  limited  to  its  use  in  connection  with  consti- 
pation; it  may  serve  in  other  capacities  as  an  aid  to  intestinal  therapeutics.'* 

It  is  frequently  desirable  for  clinical  or  experimental  purposes  to 
make  an  examination  of  the  fecal  output  which  constitutes  the  residual 
mass  from  a  certain  definite  diet.  Under  such  conditions,  it  is  customary 
to  cause  the  person  under  observation  to  ingest  some  substance,  at  the 
beginning  and  end  of  the  period  in  question,  which  shall  sufficiently 
differ  in  color  and  consistency  from  the  surrounding  feces  as  to  render 
comparatively  easy  the  differentiation  of  the  feces  of  that  period  from 
the  feces  of  the  immediately  preceding  and  succeeding  periods.  One 
of  the  most  satisfactory  methods  of  making  this  "separation"  is  by  means 
of  the  ingestion  of  a  gelatin  capsule  containing  about  0.2  gram  of  powdered 
charcoal  at  the  beginning  and  end  of  the  period  under  observation.  This 
procedure  causes  the  appearance  of  Iwo  black  zones  of  charcoal  in  the 
fecal  mass  and  thus  renders  comparatively  simple  the  differentiation  of 
the  feces  of  the  intermediate  period.  Carmine  (0.3  gram)  may  be  used  in 
a  similar  manner  and  forms  two  dark  red  zones.  Some  similar  method 
for  the  "separation  of  feces"  is  universally  practised  in  connection  with 
the  scientifically  accurate  type  of  nutrition  or  metabolism  experiment 

'  Howe  and  Hawk;  Jour.  Biol.  Ghent.,  ii,  129,  1912. 

^  Agar-agar  is  a  product  prepared  from  certain  types  of  Asiatic  sea-weed.  It  is  a  carbo- 
hydrate and  is  classified  as  a  galactan  in  tiie  polysaccharide  group. 

'Mendel:  Zent.  f.  ges.  Physiol,  u.  Path,  des  Stoffw.,  No.  17,  p.  i,  1908;  Schmidt:  Milnch. 
med.  Woch.,  52,  1970,  1905. 

^Einhorn:  Berl.  klin.  Woch.,  49,  113,  1912. 


FECES.  l8l 

which  embraces  the  collection  of  useful  data  regarding  the  income  and 
outgo  of  nitrogen,  and  other  elements. 

Among  the  macroscopical  constituents  of  the  feces  may  be  men- 
tioned the  following:  Intestinal  parasites,  undigested  food  particles, 
gall  stones,  pathological  products  of  the  intestinal  wall,  enteroliths, 
intestinal  sand,  and  objects  which  have  been  accidentally  swallowed. 

The  fecal  constituents  which  at  various  times  and  under  different 
conditions  may  be  detected  by  the  use  of  the  microscope  are  as  follows: 
Constituents  derived  from  the  food,  such  as  muscles  fibers,  connective- 
tissue  shreds,  starch  granules,  and  fat;  formed  elements  derived  from 
the  intestinal  tract,  such  as  epithelium,  erythrocytes,  and  leucocytes; 
mucus;  pus  corpuscles;  parasites  and  bacteria.  In  addition  to  the  con- 
stituents named  the  following  crystalline  deposits  may 
be  detected :  cholesterol,  soaps,  fatty  acid,  fat,  bismuth 
sulphide,  hcematoidin,  "triple  phosphate,''  Charcot- 
Leydcn  crystals,  and  the  oxalate,  carbonate,  phosphate, 
sulphate,  and  lactate  of  calcium. 

The  detection  of  minute  quantities  of  blood  in  the 

feces  ("occult  blood")  has  recently  become  a  recog-     Fig.  49.— Charcot- 
...  , .  •       r  •       1  •        1  Leyden  Crystals. 

mzed  aid  to  a  correct  diagnosis  of  certain  disorders. 

In   these    instances   the   hemorrhage  is  ordinarily   so    slight   that   the 

identification   by   means   of   macroscopical   characteristics    as    well   as 

the  microscopical  identification  through  the  detection  of  erythrocytes 

are  both  unsatisfactory  in  their  results.      Of  the  tests  given  for  the 

detection   of    "occult   blood"    the   benzidine   reaction   and    the  phenol- 

phthalein  and  aloin-turpentine  tests  (page    185)    are  probably  the  most 

satisfactory.     Since  "occult  blood"  occurs  with  considerable  regularity 

and  frequency  in  gastrointestinal  cancer  and  in  gastric  and  duodenal 

ulcer,  its  detection  in  the  feces  is  of  especial  value  as  an  aid  to  a  correct 

diagnosis  of  these  disorders. 

It  has  been  quite  clearly  shown  that  the  intestine  of  the  newly  born 
is  sterile.  However,  this  condition  is  quickly  altered  and  bacteria  may 
be  present  in  the  feces  before  or  after  the  first  ingestion  of  food.  There 
are  three  possible  means  of  infecting  the  intestine,  i.  e.,  by  way  of  the 
mouth  or  anus  or  through  the  blood.  The  infection  by  means  of  the 
blood  seldom  occurs  except  under  pathological  conditions,  thus  limiting 
the  general  infection  to  the  mouth  and  anus. 

In  infants  with  pronounced  constipation  two-thirds  of  the  dry  sub- 
stance of  the  stools  has  been  found  to  consist  of  bacteria.  In  the  stools 
of  normal  adults  probably  about  one-third  of  the  dry  substance  is  bacteria.  * 

'  Schittenhelm  and  ToUens  found  bacteria  to  comprise  42  per  cent  of  the  dry  matter. 
This  value  is,  however,  undoubtedly  too  high. 


1 82  PHYSIOLOGICAL    CHEMISTRY. 

The  average  excretion  of  dry  bacteria  in  twenty-four  hours  for  an  adult 
is  about  8  grams.  The  output  of  fecal  bacteria  has  been  found  to  undergo 
a  decrease  under  the  influence  of  water  drinking  with  meals.  ^  There  was 
also  a  decrease  in  intestinal  putrefaction,^  a  fact  which  indicates  that  at 
least  a  part  of  the  bacterial  deficit  was  made  up  of  putrefactive  organisms. 
Over  50  per  cent  of  the  total  nitrogen  of  feces  has  been  shown  to  be 
bacterial  nitrogen.^ 

Various  enzymes  have  been  detected  in  the  feces.  The  first  one  so 
demonstrated  w^as  pancreatic  amylase. "*  The  amylase  content  of  the 
feces  is  believed  to  be  an  index  of  the  activity  of  the  pancreatic  function.^ 
The  excretion  of  this  enzyme  has  been  found  to  increase  under  the 
influence  of  water  drinking  with  meals. ^  Other  enzymes  which  have 
been  found  in  the  feces  under  various  conditions  are  trypsin,  rennin, 
maltase,  sucrase,  lactase,  nuclease  and  lipase. '^ 

Some  of  the  more  important  organisms  met  with  in  the  feces  are  the 
following:^  B.  coli,  B.  lactis  aero  genes  ^  Bad.  Welchii,  B.  bijidus,  and 
coccal  forms.  Of  these  the  first  three  types  mentioned  are  gas-forming 
organisms.  The  production  of  gas  by  the  fecal  flora  in  dextrose-bouillon 
is  subject  to  great  variations  under  pathological  conditions:  alterations 
in  the  diet  of  normal  persons  will  also  cause  wide  fluctuations.  In  this 
connection  Herter  has  observed  a  marked  reduction  or  even  complete 
cessation  of  gas  production  by  the  mixed  fecal  bacteria  while  considerable 
doses  of  benzoate  were  being  given.  A  return  to  the  former  plane  of  gas 
production  followed  the  discontinuation  of  the  benzoate.®  Data  as  to  the 
production  of  gas  are  of  considerable  importance  in  a  diagnostic  way 
although  the  exact  cause  of  the  variations  is  not  yet  established.  It  should 
be  borne  in  mind  in  this  connection  that  gas  volumes  are  frequently  vari- 
able with  the  same  individual.  For  this  reason  it  is  necessary  in  every 
instance  to  follow  the  gas  production  for  a  considerable  period  of  time 
before  drawing  conclusions.  ^** 

The  nitrogen  present  in  the  feces  consists  principally  of  bacteria, 
unabsorhed  intestinal  secretions,  epithelial  cells,  mucus  material  and  food 
residues.     In  the  early  days  of  nutrition  study  the  fecal  nitrogen  was 

'  Mattill  and  Hawk:  Jour.  Am.  Chem.  Soc,  2iZ,  1999,  1911;  B'latherwick,  Shenvin  and 
Hawk:  Jour.  Biol.  Chem.,  11,  viii,  1912  (Proceedings). 

^  Hattrem  and  Hawk:  Arch.  Int.  Med.,  7,  610,  191 1;  Blatherwick,  Sherwin  and  Hawk: 
loc.  cit. 

'  MacNeal,  Latzer  and  Kerr:  Jour.  Inf.  Dis.,  6,  123,  190Q;  Mattill  and  Hawk:  Jour 
JLxp.  Med.,  14,  433,  1911;  Blatherwick  and  Hawk:  Unpublished  data. 

*  Wcgscheider:  Inaug.  Diss.,  Strassburg,  1875. 

^  Wohlgemuth:  Berl.  klin.  Woch.,  47,  3,  92,  1910. 

"Hawk:  Arch.  Int.  Med.,  8,  382,  1911. 

'  Ury:  Biochem.  Zeit.,  23,  152,  1909. 

'  Herter  and  Kendall:  Journal  of  Biological  Chemistry,  5,  283,  1908. 

"  Private  communication  from  Professor  C.  A.  Herter. 

'"  Herter  and  Kendall:  loc.  cit. 


FECES.  183 

believed  to  consist  principally  of  food  residues.  We  now  know  that  such 
residues  ordinarily  make  up  but  a  small  part  of  the  nitrogen  quota  of  the 
stools  of  normal  individuals  who  exercise  normal  mastication.^  When 
meat  has  been  "bolted,"  however,  from  1/2  gram  to  16  grams  of  macro- 
scopical  meat  residues  has  been  found  in  a  single  stool. ^  The  phrase 
"metabolic  product  nitrogen"  is  frequently  used  as  a  designation  for 
all  fecal  nitrogen  except  that  present  as  food  residues  and  bacteria. 
Bacteria  cannot  logically  be  classed  under  "metabolic"  nitrogen  since 
they  doubtless  develop  at  the  expense  of  food  nitrogen  as  well  as  at  the 
expense  of  that  in  the  form  of  intestinal  secretions.  In  the  accurate 
study  of  "  protein  utilization"^  a  correction  should  be  made  for  "  metabolic 
nitrogen."  Data  regarding  the  output  of  metabolic  nitrogen  may  be 
secured  by  determining  the  fecal  nitrogen  excretion  on  a  diet  of  proper 
energy  value  but  amtaining  no  nitrogen.*  Agar-agar  may  be  utilized 
advantageously  in  connection  with  such  a  nitrogen-free  diet. 

Feces  is  still  excreted  from  the  intestine  even  when  no  food  is  ingested. 
Carefully  conducted  fasting  experiments  have  demonstrated  this.  A  dog 
nourished  on  an  ordinary  diet  to  which  bone  ash  has  been  added  will 
excrete  a  grey  feces.  W^hen  fasted  such  an  animal  will,  after  a  few  days, 
excrete  a  small  amount  of  a  greenish-brown  mass,  containing  no  bone  ash. 
This  is  fasting  feces.  It  is  of  a  pitch-like  consistency  and  turns  black 
on  contact  with  the  air.*  Adult  fasting  men  have  been  found  to  excrete 
7-8  grams  of  feces  per  day,  the  daily  nitrogen  value  being  about  o.i  gram.® 
No  separating  medium  such  as  charcoal  or  carmine  (p.  180)  should  be  used 
in  differentiating  fasting  feces. 

In  recent  years  the  examination  of  feces  for  evidences  of  parasitism 
(detection  of  parasites  and  their  ova)  has  taken  on  an  added  importance. 
The  investigation  of  the  hookworm  has  been  particularly  developed.  (For 
methods  and  discussion  see  Bulletin  135,  Bureau  of  Animal  Industry, 
U.  S.  Department  of  Agriculture,  191 1  (M.  C.  Hall.) 

For  diagnostic  purposes  the  macroscopical  and  microscopical  exami- 
nations of  the  feces  ordinarily  yield  much  more  satisfactory  data  than 
are  secured  from  its  chemical  examination. 

'  Kerraauner:  Zeit.fiir  Biol.,  35,  316,  1897. 

-  Foster  and  Hawk:  Proceedings  of  Eighth  International  Congress  of  .Applied  Chem.,  New 
York,  September,  1912. 

^  The  percentage  of  the  ingested  protein  which  is  absorbed  from  the  intestine.  This 
may  be  calculated  by  subtracting  the  metabolic  nitrogen  from  the  total  fecal  nitrogen  and 
dividing  this  value  by  the  food  nitrogen. 

*  Tsuboi:  Zeit.fiir  Biol.,  35,  68,  1897;  Mendel  and  Fine:  Jour.  Biol.  Chem.,  ii,  5,  191 2. 

^  Howe  and  Hawk:  Jour.  Am.  Chem.Soc,  ^^,  215,  1911. 

*Howe,  Mattill  and  Hawk:  Ibid.,  ^3,  56S,  191 1. 


1 84 


PHYSIOLOGICAL   CHEMISTRY. 


Experiments  on  Feces. 


I.  Macroscopical  Examination. — If  the  stool  is  watery  pour  it 
into  a  shallow  dish  and  examine  directly.  If  it  is  firm  or  pasty  it  should 
be  treated  with  water  and  carefully  stirred  before  the  examination  for 
macroscopical  constituents  is  attempted. 

The  macroscopical  constituents  may  be  collected  very  satisfactorily 
by  means  of  a  Boas  sieve  (Fig.  50).  This  sieve  is  constructed  of  two 
easily  detachable  hemispheres  which  are  held  together  by  means  of  a 
bayonet  catch.  In  using  the  apparatus  the  feces  is  spread  out  upon  a 
very  fine  sieve  contained  in  the  lower  hemisphere  and 
a  stream  of  water  is  allowed  to  play  Upon  it  through 
the  medium  of  an  opening  in  the  upper  hemisphere. 
The  apparatus  is  provided  with  an  orifice  in  the 
upper  hemisphere  through  which  the  feces  may  be 
stirred  by  means  of  a  glass  rod  during  the  washing 
process.  After  15-30  minutes'  washing  nothing  but 
the  coarse  fecal  constitutents  remain  upon  the  sieve. 

2.  Microscopical  Examination. — Watery  stools 
should  be  placed  in  a  shallow  dish,  thoroughly  mixed, 
and  a  small  amount  removed  to  a  slide  for  examina- 
tion. Stools  of  a  firm  or  pasty  consistency  should  be 
rubbed  up  in  a  mortar  with  physiological  salt  solution 
and  a  small  portion  of  the  resulting  mixture  trans- 
ferred to  a  slide  for  examination.  In  normal  feces 
look  for  food  particles,  bacteria  and  crystalline  bodies.  In  pathological 
stools,  in  addition  to  these  substances,  look  for  animal  parasites  and 
pathological  products  of  the  intestinal  wall.     See  Fig.  47,  page  178. 

3.  Reaction. — Thoroughly  mix  the  feces  and  apply  moist  red  and 
blue  litmus  papers  to  the  surface.  If  the  stool  is  hard  it  should  be 
mixed  with  water  before  the  reaction  is  taken.  Examine  the  stool 
as  soon  after  defecation  as  is  convenient,  since  the  reaction  may  change 
very  rapidly.  The  reaction  of  the  normal  stools  of  adult  man  is  ordi- 
narily neutral  or  faintly  alkaline  to  litmus,  but  seldom  acid.  Infants' 
stools  are  generally  acid  in  reaction.  Try  the  reaction  to  Congo  red  paper. 
Also  test  tte  reaction  of  fecal  extract  to  phenolphthalein. 

4.  Starch. — If  any  imperfectly  cooked  starch-containing  food  has 
been  ingested  it  will  be  possible  to  detect  starch  granules  by  a  micro- 
scopical examination  of  the  feces.  If  the  granules  are  not  detected 
by  a  microscopical  examination,  the  feces  should  be  placed  in  an  evaporat- 
ing dish  or  casserole  and  boiled  with  water  for  a  few  minutes.  Filter 
and  test  the  filtrate  by  the  iodine  test  in  the  usual  way  (see  page  50). 


Fig. 


-Boas' 


Sieve. 


FECES.  185 

5.  Cholesterol  and  Fat. — Extract  the  dry  feces  with  ether  in  a 
Soxhlet  apparatus  (see  Fig.  136).  If  this  apparatus  is  not  available 
transfer  the  dry  feces  to  a  flask,  add  ether,  and  shake  frequently  for 
a  few  hours.  Filter  and  remove  the  ether  by  evaporation.  The  residue 
contains  cholesterol  and  the  mixed  fats  of  the  feces.  For  every  gram 
of  fat  add  about  i  1/2  gram  of  solid  potassium  hydroxide  and  25  c.c. 
of  95  per  cent  alcohol  and  boil  in  a  flask  on  a  water-bath  for  one-half 
hour,  maintaining  the  volume  of  alcohol  constant.  This  alcoholic- 
potash  has  saponified  the  mixed  fats  and  we  now  have  a  mixture  of 
soaps  and  cholesterol.  Add  sodium  chloride,  in  substance,  to  the  mixture 
and  extract  with  ether  to  dissolve  out  the  cholesterol.  Remove  the  ether 
by  evaporation  and  examine  the  residue  microscopically  for  cholesterol 
crystals.     Try  any  of  the  other  tests  for  cholesterol  as  given  on  page  272. 

6.  Blood. — Undecomposed  blood  may  be  detected  macroscopically. 
If  uncertain,  look  for  erythrocytes  under  the  microscope,  and  spectro- 
scopically  for  the  spectrum  of  oxyhaemoglobin  (see  Absorption  Spectra, 
Plate  I). 

In  case  the  blood  has  been  altered  or  is  present  in  minute  amount 
("occult  blood"),  and  cannot  be  detected  by  the  means  just  mentioned, 
the  following  tests  may  be  tried : 

{a)  Benzidine  Reaction. — Make  a  thin  fecal  suspension  using  about 
5  c.c.  of  distilled  water,  and  heat  it  to  boiling  to  render  oxidizing  enzymes 
inactive.  To  2  c.c.  of  a  saturated  solution  of  benzidine  in  glacial  acetic 
acid  add  3  c.c.  of  3  per  cent  hydrogen  peroxide  and  2-3  drops  of  the  cooled 
fecal  suspension.  A  clear  ^reg«  or  blue  color  appears  within  1-2  minutes 
in  the  presence  of  blood.  If  the  mixture  is  7iot  shaken  a  ring  of  color  will 
form  at  the  top.  Minute  traces  of  blood  are  more  easily  detected  by  the 
latter  procedure. 

{h)  Phenol phthalein  Tesl.^ — Make  a  thin  fecal  suspension  using 
about  5  c.c.  of  distilled  water.  Heat  to  boiling,'  cool  and  add  2  c.c.  of 
the  suspension  to  i  c.c.  of  the  phenolphthalein  reagent^  and  a  few  drops 
of  hydrogen  peroxide.  A  pink  or  red  color  promptly  forms  in  the  presence 
of  blood. 

(c)  Aloin-turpenline  Test. — Mix  the  stool  very  thoroughly  and  take 
about  5  grams  of  the  mixture  for  the  test.  Reduce  this  sample  to  a 
semi-fluid  mass  by  means  of  distilled  water  and  extract  very  thoroughly 

'  Boas:  Deut.  Med.   Woch.,  37,  62,   igii. 

^  Boas  suggests  using  an  ether  extract  of  the  fecal  suspension  thus  eliminating  the  necessity 
of  boiling.  However,  oxidizing  enzymes  are  the  main  sources  of  error  here  and  the  action 
is  easily  and  effectively  eliminated  by  boiling.  (See  White:  Boston  Medical  and  Surgical 
Journal,   164,  876,   iqii.) 

^Prepared  by  dissolving  1-2  grams  of  phenolphthalein  and  25  grams  of  KOH  in  100  c.c. 
of  distilled  water.  .A.dd  10  grams  of  powdered  zinc  and  heat  gently  until  the  solution  is  de- 
colorized.    Prepared  in  this  way  the  solution  will  not  deteriorate  on  standing. 


1 86  PHYSIOLOGICAL   CHEMISTRY. 

with  an  equal  volume  of  ether  to  remove  any  fat  which  may  be  present. 
Now  treat  the  extracted  feces  with  one-third  its  volume  of  glacial  acetic 
acid  and  lo  c.c.  of  ether  and  extract  very  thoroughly  as  before.  The 
acid-ether  extract  will  rise  to  the  top  and  may  be  removed. 

Introduce  2-3  c.c.  of  this  acid-ether  solution  into  a  test-tube,  add  an 
equal  volume  of  a  dilute  solution  of  aloin  in  70  per  cent  alcohol  and  2-3 
c.c.  of  ozonized  turpentine  and  shake  the  tube  gently.  If  blood  is  present 
the  entire  volume  of  fluid  ordinarily  becomes  pink  and  finally  cherry-red. 
In  some  instances  the  color  will  be  limited  to  the  aloin  solution  which 
sinks  to  the  bottom.  This  color  reaction  should  occur  within  fifteen 
minutes  in  order  to  indicate  a  positive  test  for  blood,  since  the  aloin  will 
turn  red  of  itself  if  allowed  to  stand  for  a  longer  period.  The  color  is 
ordinarily  light  yellow  in  a  negative  test.  Hydrogen  peroxide  is  not  a 
satisfactory  substitute  for  turpentine  in  the  test. 

(d)  Weber^s  Guaiac  Test. — Mix  a  little  feces  with  30  per  cent  acetic 
acid  to  form  a  fluid  mass.  Transfer  to  a  test-tube  and  extract  with  ether. 
If  blood  is  present  the  ether  will  assume  a  brownish-red  color.  Filter 
off  the  ether  extract  and  to  a  portion  of  the  filtrate  add  an  alcoholic  solu- 
tion of  guaiac  (strength  about  i :  60) ,  ^  drop  by  drop,  until  the  fluid  becomes 
turbid.  Now  add  hydrogen  peroxide  or  old  turpentine.  In  the  presence 
of  blood  a  blue  color  is  produced  (see  page  209). 

(e)  Cowie's  Guaiac  Test. — To  i  gram  of  moist  feces  add  4-5  c.c.  of 
glacial  acetic  acid  and  extract  the  mixture  with  30  c.c.  of  ether.  To  1-2 
c.c.  of  the  extract  add  an  equal  volume  of  water,  agitate  the  mixture,  intro- 
duce a  few  granules  of  powdered  guaiac  resin,  and  after  bringing  the  resin 
into  solution,  gradually  add  30  drops  of  old  turpentine  or  hydrogen 
peroxide.  A  blue  color  indicates  the  presence  of  blood.  Cowie  claims 
that  by  means  of  this  test  an  intestinal  hemorrhage  of  i  gram  can  easily 
be  detected  by  an  examination  of  the  feces. 

(/)  Acid-h(zmatin. — Examine  some  of  the  ethereal  extract  from 
Experiment  {d)  spectroscopically.  Note  the  typical  spectrum  of  acid- 
haematin  (see  Absorption  Spectra,  Plate  II). 

7.  Hydrobilirubin.  Schmidt's  Test. — Rub  up  a  small  amount  of 
feces  in  a  mortar  with  a  concentrated  aqueous  solution  of  mercuric  chloride. 
Transfer  to  a  shallow,  flat-bottomed  dish  and  allow  to  stand  6-24  hours. 
The  presence  of  hydrobilirubin  will  be  indicated  by  a  deep  red  color  being 
imparted  to  the  particles  of  feces  containing  this  pigment.  This  red  color 
is  due  to  the  formation  of  hydrobilirubin-mercury.  If  unaltered  bilirubin 
is  present  in  any  portion  of  the  feces  that  portion  will  be  green  in  color  due 
to  the  oxidation  of  bilirubin  to  biliverdin. 

'  Buckmaster  advises  the  use  of  an  alcoholic  solution  of  guaiaronic  acid  instead  of  an 
alcoholic  solution  of  guaiac  resin. 


FECES.  187 

Another  method  for  the  detection  of  hydrobilirubin  is  the  following: 
Treat  the  dry  feces  with  absolute  alcohol  acidified  with  sulphuric  acid 
and  shake  thoroughly.  The  acidified  alcohol  extracts  the  pigment  and 
assumes  a  reddish  color.  Examine  a  little  of  this  fluid  spectroscopically 
and  note  the  typical  spectrum  of  hydrobilirubin  (Absorption  Spectra, 
Plate  11). 

S.  Bilirubin/  (a)  Gmelin's  Test. — ^Place  a  few  drops  of  concentrated 
nitric  acid  in  an  evaporating  dish  or  on  a  porcelain  test-tablet  and  allow  a 
few  drops  of  the  feces  and  water  to  mix  with  it.  The  usual  play  of  colors 
of  Gmelin's  test  is  produced,  i.  e.,  green,  blue,  violet,  red,  and  yellow. 
If  so  desired,  this  test  may  be  executed  on  a  slide  and  observed  under  a 
microscope. 

(6)  HupperCs  Test. — Treat  the  feces  with  water  to  form  a  semi-fluid 
mass,  add  an  equal  amount  of  milk  of  lime,  shake  thoroughly,  and  filter. 
Wash  the  precipitate  with  water,  then  transfer  both  the  paper  and  the 
precipitate  to  a  small  beaker  or  flask,  add  a  small  amount  of  95  per  cent 
alcohol  acidified  slightly  with  sulphuric  acid,  and  heat  to  boiling  on  a 
water-bath.  The  presence  of  bilirubin  is  indicated  by  the  alcohol 
assuming  a  green  color. 

Steensma  advises  the  addition  of  a  drop  of  a  0.5  per  cent  solution  of 
sodium  nitrite  to  the  acid-alcohol  mixture  before  warming  on  the  water- 
bath.     Try  this  modification  also. 

9.  Bile  Acids. — Extract  a  small  amount  of  feces  with  alcohol  and 
filter.  Evaporate  the  filtrate  on  a  water-bath  to  drive  off  the  alcohol 
and  dissolve  the  residue  in  water  made  slightly  alkaline  with  potassium 
hydroxide.  Upon  this  aqueous  solution  try  any  of  the  tests  for  bile  acids 
given  on  page  163. 

10.  Caseinogen. — Extract  the  fresh  feces  first  with  a  dilute  solution 
of  sodium  chloride,  and  later  with  water  acidified  with  dilute  acetic 
acid,  to  remove  soluble  proteins.  Now  extract  the  feces  with  0.5  per 
cent  sodium  carbonate  and  filter.  Add  dilute  acetic  acid  to  the  filtrate 
to  precipitate  the  caseinogen,  being  careful  not  to  add  an  excess  of  the 
reagent  as  the  caseinogen  would  dissolve.  Filter  off  the  caseinogen  and 
test  it  according  to  directions  given  on  page  241.  Caseinogen  is  found 
principally  in  the  feces  of  children  who  have  been  fed  a  milk  diet.  Mucin 
would  also  be  extracted  by  the  dilute  alkali,  if  present  in  the  feces.  What 
test  could  you  make  on  the  newly  precipitated  body  to  differentiate 
between  mucin  and  caseinogen  ? 

11.  Nucleoprotein. — Mix  the  stool  thoroughly  with  water,  transfer 
to  a  flask,  and  add  an  equal  amount  of  saturated  lime  water.     Shake 

'  The  detection  of  bilirubin  in  the  feces  is  comparatively  simple  provided  it  is  not  accom- 
panied by  other  pigments.  When  other  pigments  are  present,  however,  it  is  difficult  to 
detect  the  bilirubin  and,  at  times,  may  be  found  impossible. 


l88  PHYSIOLOGICAL   CHEMISTRY. 

frequently  for  a  few  hours,  filter,  and  precipitate  the  nucleo-protein  with 
acetic  acid.     Filter  off  this  precipitate  and  test  it  as  follows: 

{a)  Phosphorus. — Test  for  phosphorus  by  fusion  (see  page  271). 

ih)  Solubility, — Try  the  solubility  in  the  ordinary  solvents. 

(c)  Protein  Color  Test. — Try  any  of  the  protein  color  tests. 

What  proof  have  you  that  the  above  body  was  not  mucin?  What 
other  test  can  you  use  to  differentiate  between  nucleoprotein  and  mucin? 

12.  Albumin  and  Globulin. — Extract  the  fresh  feces  with  a  dilute 
solution  of  sodium  chloride.  (The  preliminary  extract  from  the  prepa- 
ration of  caseinogen  (10),  above,  may  be  utilized  here.)  Filter,  and 
saturate  a  portion  of  the  filtrate  with  sodium  chloride  in  substance.  A 
precipitate  signifies  globulin.  Filter  off  the  precipitate  and  acidify  the 
filtrate  slightly  with  dilute  acetic  acid.  A  precipitate  at  this  point  signi- 
fies albumin.     Make  a  protein  color  test  on  each  of  these  bodies. 

13.  Proteose  and  Peptone. — Heat  to  boiling  the  portion  of  the 
sodium  chloride  extract  not  used  in  the  last  experiment.  Filter  off'  the 
coagulum,  if  any  forms.  Acidify  the  filtrate  slightly  with  acetic  acid  and 
saturate  with  sodium  chloride  in  substance.  A  precipitate  here  indicates 
proteose.  Filter  it  off  and  test  it  according  to  directions  given  on  page 
120.     Test  the  filtrate  for  peptone  by  the  biuret  test. 

14.  Inorganic  Constituents. — ^Prepare  a  dilute  aqueous  solution 
of  dry  feces  and  decolorize  it  by  means  of  purified  animal  charcoal. 
Make  the  following  tests  upon  the  clear  solution. 

(a)  Chlorides. — Acidify  with  nitric  acid  and  add  silver  nitrate. 

(h)  Phosphates. — Acidify  with  nitric  acid,  add  molybdic  solution, 
and  warm  gently. 

(c)  Sulphates. — Acidify  with  hydrochloric  acid,  add  barium  chloride, 
and  warm. 

15.  Konto's  Reaction  for  Indole. — Rub  up  the  stool  with  water 
to  form  a  thin  paste.  From  this  point  the  test  is  the  same  as  for  the 
detection  of  indole  in  putrefaction  mixtures  (see  page  176). 

.  16.  Schmidt's  Nuclei  Test. — This  test  serves  as  an  aid  to  the  diag- 
nosis of  pancreatic  insufficiency.  The  test  is  founded  upon  the  theory 
that  cell  nuclei  are  digestible  only  in  pancreatic  juice,  and  therefore 
that  the  appearance  in  the  feces  of  such  nuclei  indicates  insufficiency  of 
pancreatic  secretion.  The  procedure  is  as  follows:  Cubes  of  fresh  beef 
about  one-half  centimeter  square  are  enclosed  in  small  gauze  bags  and 
ingested  with  a  test  meal.  Subsequently  the  fecal  mass  resulting  from 
this  test-meal  is  examined,  the  bag  opened,  and  the  condition  of  the 
enclosed  residue  determined.  Under  normal  conditions  the  nuclei  would 
be  digested.  Therefore  if  the  nuclei  are  found  to  be  for  the  most  part 
undigested,  and  the  intervening  period  has  been  sufficient  to  permit  of 


FECES.  189 

the  full  activity  of  the  pancreatic  function  (at  least  six  hours),  it  may  be 
considered  a  sign  of  pancreatic  insufficiency. 

It  has  been  claimed  by  Steele  that  under  certain  conditions  the  non- 
digestion  of  the  nuclei  may  indicate  a  general  lowering  of  the  digestive 
power  rather  than  a  true  pancreatic  insufficiency. 

Kashiwado^  has  recently  suggested  the  use  of  stained  cell  nuclei  in 
this  test. 

17.  Einhorn's  Bead  Test.^ — This  is  a  method  for  testing  the  digestive 
function.  In  some  respects  it  is  similar  to  Sahli's  desmoid  reaction. 
The  procedure  consists  in  wrapping  the  material  under  examination 
(catgut,  fish-bone,  raw  beef,  cooked  potatoes,  thymus  gland  or  mutton 
fat,  etc.)  in  gauze  to  which  glass  beads  of  various  colors  are  attached  and 
enclosing  gauze  and  beads  in  a  gelatine  capsule.^  The  gelatine  capsule 
is  swallowed  and  the  beads  serve  to  facilitate  the  separation  of  the  gauze 
from  the  feces.  The  residue  within  the  gauze  is  then  examined.  If 
beads  appear  in  much  less  than  24  hours  an  accelerated  motility  is  indi- 
cated, whereas  an  interval  of  48  hours  or  over  elapsing  indicates  retarded 
motility.  If  gastric  function  alone  is  to  be  studied  silk  threads  are 
attached  to  the  beads  and  the  latter  are  withdrawn  and  examined  before 
they  have  passed  into  the  intestine. 

18.  "Separation"  of  Feces. — In  order  to  become  familiar  with  the 
method  ordinarily  utilized  in  metabolism  experiments  to  differentiate  the 
feces  which  corresponds  to  the  food  ingested  during  any  given  interval, 
and  at  the  same  time  to  secure  data  as  to  the  length  of  time  necessary 
for  ingested  substances  to  pass  through  the  alimentary  tract  proceed  as 
follows:  Just  before  one  of  the  three  meals  of  the  day  ingest  a  gelatine 
capsule  (No.  00)  containing  0.2-0.3  ^^  ^  gram  of  carmine  or  charcoal. 
Make  an  inspection  of  all  stools  subsequently  dropped  and  note  the  time 
interval  elapsing  between  the  ingestion  of  the  capsule  and  the  appearance 
of  its  contents  in  the  feces.  Under  normal  conditions  this  period  is  ordi- 
narily 24  hours. 

19.  Quantitative  Determination  of  Fecal  Amylase  (The  Author's* 
Modification  of  Wohlgemuth's^  Method). — Weigh  accurately  about  2 
grams  of  fresh  feces  into  a  mortar,"  add  8  c.c.  of  a  phosphate-chloride 
solution  (o.i  mol  dihydrogen  sodium  phosphate  and  0.2  mol  disodium 
hydrogen  phosphate  per  liter  of  i  per  cent  sodium  chloride),  2  c.c.  at  a 
time,  rubbing  the  feces  mixture  to  a  homogeneous  consistency  after  each  ad- 

*  Kashiwado:  Dent.  Arch.  Klin.  Med.,  104,  584,  1911. 

''Einhorn:  The  Post-Graduate,  May  1912:  Boas'  Arch.,  12,  26,  1906;  13,  35,  1907;  Ibid, 
475;  15.  part  2,  1909. 

'  Ordinarily  two  substances  are  attached  to  each  bead,  three  beads  tied  together  and 
enclosed  in  one  capsule.     Test  capsules  may  be  obtained  from  Eimei  and  Amend,  New  York. 

*  Hawk:  .Arch.  Int.  Med.,  8,  552,  1911. 

'  Wohlgemuth:  Berl.  klin.  Woch..  47,  3,  92,  1910;  also  see  chapter  on  Enzymes,  this  book. 
"  Duplicate  determinations  should  be  made. 


190 


PHYSIOLOGICAL    CHEMISTRY. 


dition  of  the  extraction  medium.  Permit  the  mixture  to  stand  at  room  tem- 
perature for  a  half-hour  with  frequent  stirring.  We  now  have  a  neutral 
fecal  suspension.  Transfer  this  suspension  to  a  15  c.c.  graduated  centrifuge 
tube,  being  sure  to  wash  the  mortar  and  pestle  carefully  with  the  phosphate- 
chloride  solution  and  add  all  washings  to  the  suspension  in  the  centrifuge 
tube.  The  suspension  is  now  made  up  to  the  15  c.c.  mark  with  the 
phosphate-chloride  solution  and  centrifugated  for  a  fifteen-minute  period, 
or  longer  if  necessary,  to  secure  satisfactory  sedimentation.  At  this  point, 
read  and  record  the  height  of  the  sediment  column.  Remove  the  super- 
natant Hquid  by  means  of  a  bent  pipette,  transfer  it  to  a  50  c.c.  volumetric 
flask  and  dilute  it  to  the  50  c.c.  mark  with  the  phosphate-chloride  solution. 
Mix  the  fecal  extract  thoroughly  by  shaking  and  determine  its  amylolytic 
activity.  For  this  purpose  a  series  of  six  graduated  tubes  is  prepared,  con- 
taining volumes  of  the  extract  ranging  from  2.5  c.c.  to  0.078  c.c.  Each  of 
the  intermediate  tubes  in  this  series  will  thus  contain  one-half  as  much 
fluid  as  the  preceding  tube.  Now  make  the  contents  of  each  tube  2.  5 
c.c.  by  means  of  the  phosphate-chloride  solution  in  order  to  secure  a  uni- 
form electrolyte  concentration.  Introduce  5  c.c.  of  a  i  per  cent  soluble 
starch  solution^  and  three  drops  of  toluol  into  each  tube,  thoroughly  mix 
the  contents  by  shaking,  close  the  tubes  by  means  of  stoppers  and  place 
them  in  an  incubator  at  38°  C.  for  twenty-four  hours.  At  the  end  of  this 
time  remove  the  tubes,  fill  each  to  within  half  an  inch  of  the  top  with  ice- 
water,  add  one  drop  of  tenth-normal  iodin  solution,  thoroughly  mix  the 
contents  and  examine  the  tubes  carefully  with  the  aid  of  a  strong  light. 
Select  the  last  tube  in  the  series  which  shows  entire  absence  of  blue  color, 
thus  indicating  that  the  starch  has  been  completely  transformed  into  dex- 
trin and  sugar,  and  calculate  the  amylolytic  activity  on  the  basis  of  this 
dilution.  In  case  of  indecision  between  two  tubes,  add  an  extra  drop  of 
the  iodin  solution  and  observe  them  again. ^ 

*  In  preparing  the  i  per  cent  solution,  the  weighed  starch  powder  should  be  dissolved 
in  cold  distilled  water  in  a  casserole  and  stirred  until  a  homogeneous  suspension  is  obtained. 
The  mixture  should  then  be  heated  with  constant  stirring,  until  it  is  clear.  This  ordinarily 
takes  from  eight  to  ten  minutes.  A  slightly  opaque  solution  is  thus  obtained,  which  should 
be  cooled  and  made  up  to  the  proper  volume  before  using. 

''■  Theoretically  we  would  expect  the  colors  to  range  from  a  light  yellow  to  a  dark  blue, 
with  red  tubes  holding  an  intermediate  position  in  the  series.  This  color  sequence  does  often 
occur,  but  its  occurrence  is  far  from  universal.  Many  times  the  first  tubes  in  the  series,  i.  e., 
those  containing  the  largest  quantities  of  the  fecal  extract,  will  exhibit  a  bluish  cast  of  color 
which  should  not  be  confused  with  the  starch  color  reaction.  When  these  blue  tubes  are 
present,  they  are  generally  followed  by  yellow,  red  and  blue  tubes  in  order,  the  final  blue  tube, 
of  course,  being  the  regulation  starch  reaction.  Occasionally  greenish  colors  will  be  obtained 
to  the  left  of  the  red  color.  It  also  sometimes  happens  that  it  is  somewhat  difficult  to  determine 
in  which  tube  to  the  right  of  the  red  color  the  starch  blue  color  is  first  detected,  unless  the 
tube  be  examined  carefully  before  a  strong  light.  In  every  instance,  however,  when  these  blue 
anri  green  colors  are  observed,  it  is  noted  that  tubes  possessing  the  true  dextrin  red  color  are 
always  present  between  these  tubes  and  the  tubes  possessing  the  true  starch  blue  color.  It 
is  evident,  therefore,  that  these  bluish  tints  in  the  tubes  to  the  left  of  the  dextrin  color  cannot 
be  due  to  the  presence  of  starch.  The  cause  of  the  blue  color  reaction  in  the  first  tubes  of 
the  series  has  not  been  ascertained  as  yet. 


FECES.  191 

The  amylolytic  value,  Df,  of  a  given  stool,  may  be  expressed  in  temrs 
of  I  c.c.  of  the  sediment  obtained  by  centrifugation  as  above  described. 
For  example,  if  it  is  found  that  0.31  c.c.  of  the  phosphate-chloride  extract 
of  the  stool  acting  at  38°  C.  for  twenty-four  hours  completely  transformed 
the  starch  in  5  c.c.  of  a  i  per  cent  starch  solution,  then  we  would  have  the 
following  proportion: 

0.31  :  5  (c.c.  starch)  : :  i  (c.c.  extract):  X 

The  value  of  X  in  this  case  is  16.  i,  which  means  that  i  c.c.  of  the  fecal 
extract  possesses  the  power  of  completely  digesting  16.  r  c.c.  of  a  i  per  cent 
starch  solution  in  twenty-four  hours  at  38°  C. 

Inasmuch  as  stools  vary  so  greatly  as  to  water  content,  it  is  essential 
to  an  accurate  comparison  of  stools  that  such  comparison  be  made  on  the 
basis  of  the  solid  matter.  Supposing,  for  example,  that  in  the  above 
determination  we  had  6.2  c.c.  of  sediment.  Since  the  supernatant  fluid 
was  removed  and  made  up  to  50  c.c.  before  testing  its  amylolytic  value, 
it  is  evident  that  i  c.c.  of  this  sediment  is  equivalent  to  8.1  c.c.  of  extract. 
Therefore,  in  order  to  derive  the  amylolytic  value  of  i  c.c.  of  sediment, 
we  must  multiply  the  value  (16.  i)  as  obtained  above  for  the  extract,  by  8.1. 
This  yields  130.4  and  enables  us  to  express  the  activity  as  follows: 

^r38  c 

Df^       =130.4 
24  h 

The  above  method  of  calculation  is  that  suggested  by  Wohlgemuth.  In 
case  time  and  facilities  permit  of  the  determination  of  the  moisture  con- 
tent of  the  feces,  it  is  much  more  accurate  and  satisfactory  to  place  the 
amylolytic  values  of  the  stools  on  a  "gram  of  dry  matter"  basis.  The 
amylolytic  values  of  the  stools  are  expressed  as  the  number  of  cubic  centi- 
meters of  I  per  cent  starch  solution  which  the  amylase  content  of  i  gram 
of  dry  feces  is  capable  of  digesting. 

20.  Quantitative  Determination  of  Fecal  Bacteria. ' — The  method 
is  a  simplification  of  MacNeal's  adaptation  of  the  Strasburger  procedure.' 
"About  two  grams  of  feces  are  accurately  weighed  and  placed  in  a  50  c.c. 
centrifuge  tube.  To  the  feces  in  the  tube  a  few  drops  of  0.2  per  cent 
hydrochloric  acid  are  added,  and  the  material  is  mixed  to  a  smooth  paste 
by  means  of  a  glass  rod.  Further  amounts  of  the  acid  are  added  with  con- 
tinued crushing  and  stirring  until  the  material  is  thoroughly  suspended. 
The  tube  is  then  whirled  in  the  centrifuge  at  high  speed  for  one  half  to  one 
minute.  The  suspension  is  found  sedimented  into  more  or  less  definite 
layers,  the  uppermost  of  which  is  fairly  free  from  the  larger  particles. 
The  upper  and  more  liquid  portion  of  the  suspension  is  now  drawn  off  by 

'Mattill  and  Hawk:  Jour.  Exp.  Med.,  14,  433,  1911. 

'  MacNeal,  Latzer  and  Kerr,  Jour.  Inf.  Dis.,  6,  123,  1909. 


192  PHYSIOLOGICAL   CHEMISTRY, 

means  of  a  pipette  and  transferred  to  a  beaker,  ^  The  sediment  remaining 
in  the  tube  is  again  rubbed  up  with  the  glass  rod  with  the  addition  of 
further  amounts  of  dilute  acid,  and  again  centrifugalized  for  one  half  to 
one  minute.  The  supernatant  liquid  is  pipetted  off  and  added  to  the 
first,  the  same  pipette  being  used  for  the  one  determination  throughout.^ 
A  third  portion  of  the  dilute  acid  is  then  added  to  the  sediment,  which  is 
again  mixed  by  stirring  and  again  centrifugalized.  All  the  washings  are 
added  to  the  first  one,  and  during  the  process  care  is  taken  to  wash  the 
material  from  the  walls  and  mouth  of  the  centrifuge  tube  down  into  it. 
Finally,  when  the  sediment  is  sufficiently  free  from  bacteria,  the  various 
remaining  particles  are  visibly  clean,  and  the  supernatant  liquid  after 
centrifugalization  remains  almost  clear.  This  is  removed  to  the  beaker  in 
which  are  now  practically  all  the  bacteria  present  in  the  original  portion 
of  feces,  together  with  some  solid  matter  not  yet  separated.  In  the  centri- 
fuge tubes  there  is  a  considerable  amount  of  bacteria-free  solid  matter. 

The  suspension  is  now  transferred  to  the  same  centrifuge  tube,  centrif- 
ugalized for  a  minute,  and  the  supernatant  hquid  transferred  to  a  clean 
beaker  by  means  of  the  same  pipette.  The  tube  is  then  refilled  from  the 
first  beaker  and  thus  all  the  suspension  centrifugalized  a  second  time. 
The  beaker  is  finally  carefully  washed  with  the  aid  of  a  rubber-tipped 
glass  rod,  the  second  sediment  in  the  centrifuge  tube  is  washed  free  of 
bacteria  by  means  of  this  wash  water  and  by  successive  portions  of  the 
dilute  acid,  and  the  supernatant  liquid  after  centrifugalization  is  added  to 
the  contents  of  the  second  beaker.  The  second  clean  sediment  is  added 
to  the  first.  The  bacterial  suspension  now  in  the  second  beaker  is  again 
centrifugalized  in  the  same  way  and  a  third  portion  of  bacteria-free 
sediment  is  separated.  Frequently  a  fourth  serial  centrifugalization  is 
performed — always  if  the  third  sediment  is  of  appreciable  quantity. 
At  all  stages  of  the  separation,  small  portions  of  the  dilute  hydro- 
chloric acid  are  used,  so  that  the  final  suspension  shall  not  be  too  vo- 
luminous. Ordinarily  it  amounts  to  125  to  200  c.c.  At  the  same  time, 
the  final  amount  of  fluid  should  not  be  too  small,  as  shown  by 
Ehrenpfordt,^  because  the  viscosity  accompanying  increased  concentration 
prevents  proper  arid  complete  sedimentation. 

To  the  final  bacterial  suspension  an  equal  volume  of  alcohol  is  added 
and  the  beaker  set  aside  to  concentrate.  A  water  bath  at  50°  to  60°  C.  is 
very  satisfactory.  After  two  or  three  days,  when  the  liquid  is  concen- 
trated to  about  50  c.c,  the  beaker  is  removed  and  about  200  c.c.  of  alcohol 

'  A  25  c.c.  pipette  is  the  most  satisfactory  size;  to  facilitate  observation,  the  delivery  tube 
is  bent  near  the  bulb  to  an  angle  of  about  120  degrees. 

^  A  convenient  support  for  the  pipettes  is  a  wire  spring  on  a  glass  base,  such  as  is  used  on  a 
desk,  for  pen-holders.  The  delivery  tube,  just  where  it  is  bent,  is  inserted  between  the  wires, 
and  any  liquid  not  delivered  collects  in  the  bend  of  the  tube. 

'  Ehrenpfordt:  Zeil.  exp.  Path.  Ther.,  7,  455,  iqoq. 


FECES.  193 

are  added.  The  beaker  is  covered  and  allowed  to  stand  at  room  tempera- 
ture for  twenty-four  hours.  At  the  end  of  this  time  the  bacterial  sub- 
stance is  generally  settled,  so  that  most  of  the  clear  supernatant  liquid,  of 
dark,  brown  color,  can  be  directly  siphoned  off  without  loss  of  solid  matter. 
The  remainder  is  then  transferred  to  centrifuge  tubes,  centrifugalized, 
and  the  remaining  clear  liquid  pipetted  off.  ^  The  sediment  consists  of  the 
bodies  of  the  bacteria,  and  is  transferred  to  a  Kjeldahl  flask  for  nitrogen 
determination.  "This  is  the  bacterial  nitrogen.  Where  a  determination 
of  bacterial  dry  substance  is  desired,  the  sediment  of  bacteria  is  extracted 
by  absolute  alcohol  and  ether  in  succession,  transferred  to  a  weighed 
porcelain  crucible,  and  dried  at  102°  C.  to  constant  weight.  This  dried 
sample  is  then  used  in  the  nitrogen  determination.  Our  procedure  differs 
from  that  of  MacNeal  in  that  the  bacterial  dry  matter  is  not  determined. 
A  saving  of  about  seven  days'  time  and  of  considerable  labor  is  accom- 
plished by  this  omission. 

Inasmuch  as  it  has  been  shown  by  various  investigators  that  such 
bacteria  as  are  present  in  the  feces  contain  on  the  average  about  i  r  per 
cent  of  nitrogen,  the  values  for  bacterial  nitrogen  as  determined  by  our 
method  may  conveniently  serve  as  a  basis  for  the  calculation  of  the  actual 
output  of  bacterial  substance. 

'  In  more  recent  work  (see  Blatherwick  and  Hawk:  unpublished)  it  has  been  found 
advantageous  to  centrifugalize  with  alcohol  and  ether  in  succession  before  transferring  the 
bacterial  cells  to  Kjeldahl  flasks. 


13 


CHAPTER  XII. 
BLOOD  AND  LYMPH. 

Blood  is  composed  of  four  types  of  form-elements  (erythrocytes  or 
red  blood  corpuscles,  leucocytes  or  white  blood  corpuscles,  blood  plates 
or  plaques  and  blood  dust  or  hasmoconien)  held  in  suspension  in  a  fluid 
called  blood  plasma.  These  form-elements  compose  about  60  per  cent  of 
the  blood,  by  weight.  Ordinarily  blood  is  a  dark  red  opaque  fluid  due  to 
the  presence  of  the  red  blood  corpuscles,  but  through  the  action  of  certain 
substances,  such  as  water,  ether,  or  chloroform,  it  may  be  rendered  trans- 
parent. Blood  so  altered  was  formerly  said  to  be  laked.  The  term 
hemolysis  is  now  used  in  this  connection  and  substances  which  cause  such 
action  are  spoken  of  as  hcEmolytic  agents.  The  haemolytic  process  is 
simply  a  liberation  of  the  haemoglobin  from  the  stroma  of  the  red  blood 
corpuscle.  Normal  blood  is  alkaline  in  reaction^  to  litmus,  the  alkalinity 
being  due  principally  to  sodium  carbonate  and  phosphate.  The  specific 
gravity  of  the  blood  of  adults  ordinarily  varies  between  1.045  ^^^  '^•°1S- 
It  varies  somewhat  with  the  sex,  the  blood  of  males  having  a  rather  higher 
specific  gravity  than  that  of  females  of  the  same  species.  Under  patholog- 
ical conditions  also  the  density  of  the  blood  may  be  very  greatly  altered. 
The  freezing-point  {A)  of  normal  blood  is  about  — 0.56°  C.  Variations 
between  ■ — 0.51°  and  0.62°  C.  may  be  due  entirely  to  dietary  conditions, 
but  if  any  marked  variation  is  noted  it  can  in  most  cases  be  traced  to  a 
disordered  kidney  function.  The  total  amount  of  blood  in  the  body  has 
been  variously  estimated  at  from  one-twelfth  to  one-fourteenth  of  the 
body  weight.  Perhaps  1/13.5  is  the  most  satisfactory  figure.  Abderhal- 
den  and  Schmidt^  have  recently  suggested  a  unique  method  for  the 
determination  of  this  value.  It  is  based  upon  the  change  in  the  optical 
activity  of  the  blood  upon  injection  of  a  body  of  known  optical  activity, 
such,  for  example,  as  dextrin. 

Among  the  most  important  constituents  of  blood  plasma  are  the  four 
protein  bodies,  fibrinogen,  nudeo protein,  serum  globulin  (euglobulin  and 
pseudo-globulin)  and  serum  albumin.  Plasma  contains  about  8.2  per  cent 
of  solids  of  which  the  protein  constituents  named  above  constitute  approxi- 
mately 84  per  cent  and  the  inorganic  constituents  (mainly  chlorides, 
phosphates  and  carbonates)  approximately  10  per  cent.  Among  the 
inorganic  constituents  sodium  chloride  predominates.     To  prevent  coagu- 

'  Recently  it  has  been  shown  by  physico-chemical  methods  that  the  blood  is  in  reality 
neutral  in  reaction. 

*  Abderhalden  and  Schmidt:  Zeit.  physiul.  chem.,  66,  120,  1910. 

194 


BLOOD   AND    LYMPH.  I95 

lation,  blood  plasma  is  ordinarily  studied  in  the  form  of  an  oxalated  or 
salted  plasma.  The  former  may  be  obtained  by  allowing  the  blood  to 
flow  from  an  opened  artery  into  an  equal  volume  of  0.2  per  cent  ammo- 
nium oxalate  solution,  whereas  in  the  preparation  of  a  salted  plasma  10 
per  cent  sodium  chloride  solution  may  be  used  as  the  diluting  fluid. 

Fibrinogen  is  perhaps  the  most  important  of  the  protein  constituents 
of  the  plasma.^  It  is  also  found  in  lymph  and  chyle  as  well  as  in  certain 
exudates  and  transudates.  Fibrinogen  possesses  the  general  properties 
of  the  globulins,  but  differs  from  serum  globulin  in  being  precipitated 
upon  half-saturation  with  sodium  chloride.  In  the  process  of  coagulation 
of  the  blood  the  fibrinogen  is  transformed  into  fibrin.  This  fibrin  is  one 
of  the  principal  constituents  of  the  ordinary  blood  clot. 

The  nucleoprotein  of  blood  possesses  many  of  the  characteristics  of 
serum  globulin.  In  common  with  this  body  it  is  easily  soluble  in  sodium 
chloride,  and  is  completely  precipitated  from  its  solutions  upon  saturation 
with  magnesium  sulphate.  It  is  much  less  soluble  in  dilute  acetic  acid 
than  serum  globuHn,  and  its  solutions  coagulate  at  65°-69°  C. 

The  body  formerly  called  serum  globulin  is  probably  not  an  indi- 
vidual substance.  Recent  investigations  seem  to  indicate  that  it 
may  be  resolved  into  two  individual  bodies  called  euglohulin  and  pseudo- 
globidm.  The  euglobulin  is  practically  insoluble  in  water  and  may  be 
precipitated  in  the  presence  of  28-36  per  cent  of  saturated  ammonium 
sulphate  solution.  The  pseudoglobulin,  on  the  contrary,  is  soluble  in 
water  and  is  only  precipitated  by  ammonium  sulphate  in  the  presence  of 
from  36  to  44  per  cent  of  saturated  ammonium  sulphate  solution. 

In  common  with  serum  globulin  the  body  known  as  serum  albumin 
seems  also  to  consist  of  more  than  a  single  individual  substance.  The  so- 
called  serum  albumin  may  be  separated  into  at  least  two  distinct  bodies, 
one  capable  of  crystallization,  the  other  an  amorphous  body.  The  solution 
of  either  of  these  bodies  in  water  gives  the  ordinary  albumin  reactions. 
The  coagulation  temperature  of  the  serum  albumin  mixture  as  it  occurs  in 
serum  or  plasma  varies  from  70°  to  85°  C.  according  to  the  reaction  of  the 
solution  and  its  content  of  inorganic  material.  Serum  albumin  differs 
from  egg  albumin  in  being  more  laevorotatory,  in  being  rendered  less 
insoluble  by  alcohol,  and  in  the  fact  that  when  precipitated  by  hvdro- 
chloric  acid  it  is  more  easily  soluble  in  an  excess  of  the  reagent. 

When  blood  coagulates  and  the  usual  clot  forms,  a  light  yellow  fluid 
exudes.  This  is  blood  serum.  It  differs  from  blood  plasma  in  containing 
a  large  amount  oi  fibrin  ferment,  a  body  of  great  importance  in  the  coagu- 
lation of  the  blood,  and  also  in  possessing  a  lower  protein  content.  The 
protein  material  present  in  plasma  and  not  found  in  serum  is  the  fibrin- 
ogen which  is  transformed  into  fibrin  in  the  process  of  coagulation  and 


196  PHYSIOLOGICAL   CHEMISTRY. 

removed.  The  specific  gravity  of  the  serum  of  human  blood  varies 
between  1.026  and  1.032.  If  blood  be  drawn  into  a  vessel  and  allowed 
to  remain  \^dthout  stirring  or  agitation  of  any  sort  the  major  portion  of  the 
red  corpuscles  will  sink  away  from  the  upper  surface,  causing  this  portion 
of  the  clot  to  assume  a  lighter  color  due  to  the  predominance  of  leuco- 
cytes.    This  light  colored  portion  of  the  clot  is  called  the  "buffy  coat." 

Beside  the  protein  constituents  already  mentioned,  other  bodies  which 
are  found  in  both  the  plasma  and  serum  are  the  following:  Sugar  (dex- 
trose), fat,  enzymes,  lecithin,  cholesterol  and  its  esters,  gases,  coloring- 
matter  (lutein  or  lipochrome)  and  mineral  substances.  In  addition  to 
these  bodies  the  following  substances  have  been  detected  in  normal  human 
blood:  Creatine,  carhamic  acid,  hippuric  acid,  paralactic  acid,  urea  and 
uric  acid  (urates).  Some  of  the  pathological  constituents  of  blood  are 
proteoses,  leucine,  tyrosine  and  other  amino  acids,  biliary  constituents  and 
purine  bodies. 

By  waters^  reports  the  presence  of  a  glycoprotein  in  blood  serum. 
This  he  has  termed  seromucoid. 

There  has  been  considerable  controversy  regarding  the  form  of  the 
erythrocytes  or  red  blood  corpuscles  of  human  blood.  It  is  claimed 
by  some  investigators  that  the  cells  are  bell-shaped  or  cup-shaped.  As 
the  erythrocytes  occur  normally  in  the  circulation,  however,  they  are  prob- 
ably thin,  non-nucleated,  biconcave  discs.  When  examined  singly  under 
the  microscope,  they  possess  a  pale  greenish-yellow  color  (see  Plate  IV, 
opposite),  whereas  when  grouped  in  large  masses  a  reddish  tint  is  noted. 

The  blood  of  most  mammals  contains  erythrocytes  similar  in  form  to 
those  of  human  blood.  In  the  blood  of  birds,  fishes,  amphibians  and 
reptiles  the  erythrocytes  are  ordinarily  more  or  less  elliptical,  biconvex 
and  possess  a  nucleus.  The  erythrocytes  vary  in  size  with  the  different 
animals.  The  average  diameter  of  the  erythrocytes  of  blood  from  various 
species  is  given  in  the  following  table  :^ 

Elephant ^^as  of  an  inch. 

Guinea-pig x-h  %  o^  an  inch. 

Man V .  .  ^3^5  u  of  an  inch. 

Monkey s-h-x  of  an  inch. 

Dog -irhi  of  an  inch. 

Rat aoS i  of  an  inch. 

Rabbit ^/r,  s  of  an  inch. 

Mouse ^-V;i-  of  an  inch. 

Lion ii-i ;f  of  an  inch. 

Ox .f  .jS  9  of  an  inch. 

Horse 52^4;:^  of  an  inch. 

Pig j-jV,  „  of  an  inch. 

Cat ^/f  a  of  an  inch. 

Sheep -4 oVa  of  an  inch. 

Goat Tj-ji^ ,,  of  an  inch. 

Musk-deer Tslsr,  of  an  inch. 

'  Bywaters:  Biochemische  Zeilschrift,  15,  322,  1909. 

*  Wormley's  Micro-Chemistry  of  Poisons,  second  edition,  p.  733. 


I'LATK  IV. 


Normal  Erythrocytes  and  Leucocytes. 


BLOOD   AND    LYMPH.  197 

The  erythrocytes  from  whatever  source  obtained,  consist  essentially 
of  two  parts,  the  stroma  or  protoplasmic  tissue  and  its  enclosed  pigment, 
hcemoglohin.  For  human  blood  the  number  of  erythrocytes  present  in  the 
fluid  as  obtained  from  well-developed  males  in  good  physical  condition  is 
about  5,500,000  per  cubic  millimeter.  ^  The  normal  content  of  the  blood 
of  adult  females  is  from  4,000,000  to  4,500,000  per  cubic  millimeter. 
The  number  oi  erythrocytes  varies  greatly  under  different  conditions. 
For  instance  the  number  may  be  increased  after  the  transfusion  of  blood 
of  the  same  species  of  animal;  by  residing  in  a  high  altitude;  or  as  a  result 
of  strenuous  physical  exercise  continued  over  a  short  period  of  time.  An 
increase  is  also  noted  in  starvation;  after  partaking  of  food;  after  cold  or 
hot  baths;  after  massage,  as  well  as  after  the  administration  of  certain 
drugs  and  accompanying  certain  diseases,  such  as  cholera,  diarrhoea, 
dysentery  and  yellow  atrophy  of  the  liver.  A  decrease  in  the  number 
occurs  in  the  different  forms  of  anaemia.  The  number  has  been  known 
to  increase  to  7,040,000  per  cubic  millimeter  as  a  result  of  physical  exercise, 
while  11.000,000  per  cubic  millimeter  have  been  noted  in  cases  of  poly- 
cythaemia  and  increases  nearly  as  great  in  cyanosis.  The  number  has 
been  known  to  decrease  to  500,000  per  cubic  millimeter  or  lower  in  per- 
nicious anaemia. 

Erythrocytes  possess  the  property,  when  properly  treated,  of  "clump- 
ing" together  in  masses  and  precipitating,  producing  so-called  aggluti- 
nation. Cells  other  than  erythrocytes  {e.  g.,  bacteria)  possess  this  property. 
When  spoken  of  in  connection  with  the  blood  such  action  is  termed 
hcemagglutination.  A  substance  w'hich  vAW  bring  about  haemagglutination 
is  said  to  contain  hcEmagglutinins.  These  haemagglutinins  are  particu- 
larly abundant  in  the  vegetable  kingdom.^  For  a  demonstration  of 
haemagglutination  see  page  208. 

Oxyhsemoglobin,  the  coloring  matter  of  the  blood,  is  a  conjugated 
protein.  Through  treatment  with  hydrochloric  acid  it  may  be  split  into 
a  protein  body  called  globin,  and  hcemochromogen,  an  iron-containing 
pigment.  The  latter  body  is  rapidly  transformed  into  hcEniatin  in  the 
presence  of  oxygen,  and  this  in  turn  gives  place  to  haematin- hydrochloride 
or  Immin  (Figs.  59  and  60,  page  211).  The  pigment  of  arterial  blood  is 
for  the  most  part  loosely  combined  with  oxygen  and  is  termed  oxyhsercvo- 
globin,  whereas  the  pigment  of  venous  blood  is  principally  haemoglobin 
(so-called  reduced  haemoglobin).  Oxyhaemoglobin  is  the  oxygen-carrier 
of  the  body  and  belongs  to  the  class  of  bodies  known  as  respiratory  pig- 

*  This  statement  is  based  upon  observations  made  upon  the  blood  of  athletes  in  training. 
See  Hawk:  Anter.  Jottr.  Physiol  1904.  It  is  generally  stated  in  text-books  that  the  blood 
of  males  contains  about  5,000,000  per  cubic  millimeter. 

*  Mendel:  Archivio  di  fisiologia,  7,  168,  1909;  also  Schneider:  Journal  0/ Biological  Chem., 
II,  47.  1912. 


198 


PHYSIOLOGICAL   CHEMISTRY. 


Fig.  51. — Oxyhemoglobin  Crystals  from  Blood  of  the  Guinea-pig. 
Reproduced  from  a  micro-photograph  furnished  by  Prof.  E.  T.  Reichert,  of  the  University 

of  Pennsylvania. 


V' 


>1^ 


Fig.  52. — Oxyhemoglobin  Crystals  from  Blood  of  the  Rat. 
Reproduced  from  a  micro-photograph  furnished  by  Prof.  E.  T.  Reichert,  of  the  University 

of  Pennsylvania. 


BLOOD  AND    LYMPH. 


199 


Fig.  53. — OxYH>EMOGLOBiN  Crystals  from'Blood  of  the  Horse. 
Reproduced  from  a  micro-photograph  furnished  by  Prof.  E.  T.  Reichert,  of  the  University 

of  Pennsylvania. 


i> 


Fig.  54. — Oxyhemoglobin  Crystals  from  Blood  of  the  Squirrel. 
Reproduced  from  a  micro-photograph  furnished  by  Prof.  E.  T.  Reichert,  of  the  University 

of  Pennsylvania. 


200 


PHYSIOLOGICAL   CHEMISTRY. 


di=«^" 


Fig.  55. — Oxyhemoglobin  Crystals  from  Blood  of  the  Dog. 
Reproduced  from  a  micro-photograph  furnished  by  Prof.  E.  T.  Reichert,  of  the  University 

of  Pennsylvania. 


Fig.    56. — OXYHiEMOGLOBIN   CRYSTALS   FROM    BlOOD   OF  THE   CaT. 

Ref)roduced  from  a  micro-photograph  furnished  by  Prof.  E.  T.  Reichert,  of  the  University 

of  Pennsylvania. 


BLOOD   AND    LYMPH. 


20I 


Fig.  57. — OxYH,«MOGLOBiN  Crystals  from  Blood  of  the  Necturus. 
Reproduced  from  a  micro-photograph  furnished  by  Prof.  E.  T.  Reichert,  of  the  University 

of  Pennsylvania. ' 


ments.  It  is  held  within  the  stroma  of  the  erythrocyte.  The  reduction 
of  oxyhaemoglobin  to  form  haemoglobin  (so-called  reduced  haemoglobin) 
occurs  in  the  capillaries.  O.xyhaemoglobin  may  be  crystallized  and  a 
specific  form  of  crystal  obtained  from  the  blood  of  each  individual  species 
(see  Figs.  51  to  57,  pages  198  to  201).  This  fact  seems  to  indicate  that 
there  are  many  varieties  of  oxyhaemoglobin.  The  interesting  findings 
of  Reichert  and  Brown  are  of  great  value  in  this  connection.  These 
investigators  prepared  oxyhaemoglobin  crystals  from  the  blood  of  over 
mic  hundred  species  of  animal  and  subsequently  studied  the  character- 
istics of  the  crystals  very  minutely  from  the  standpoint  of  crystallography. 
Their  findings  may  prove  of  importance  from  the  standpoint  of  heredity 
and  the  origin  of  species.     They  emphasize  the  following  facts: 

1.  Crystals  from  all  species  of  a  certain  genus  have  certain  charac- 
teristics in  general.  Crystals  from  different  genera,  however,  exhibit 
marked  differences  in  system,  axial  ratios,  etc. 

2.  Crystals  of  different  species  of  a  genus  may  generally  be  differen- 
tiated by  difference  in  the  angles. 

3.  The  oxyhaemoglobin  of  some  species  crystallizes  in  several  types 
of  crystals  in  the  same  preparation.  Generally  the  crystals  first  formed 
belong  to  a  system  of  a  lower  grade  of  symmetry  than  those  formed 
later.  When  such  dift'erent  types  of  crystals  occur  they  may  be  arranged 
in  isomorphous  series. 

4.  Certain  definite  angles  recur  in  the  crystals  from  the  blood  of 

^  The  micro- photographs  of  oxyhaemoglobin  (see  pages  108-201)  and  h.nemin  (see  page 
211)  are  reproduced  through  the  courtesy  of  Professors  E.  T.  Reichert  and  Amos  P.  Brown, 
of  the  Universitv  of  Pennsylvania,  who  are  investigating  the  crystalline  forms  of  biochemic 
substances. 


202  PHYSIOLOGICAL   CHEMISTRY. 

various  species  of  animal,  although  the  zoological  connection  may  be 
remote  and  the  crystals  belong  to  different  systems. 

5.  The  constant  recurrence  of  certain  types  of  "twinning"  in  all 
the  crystalline  forms  was  observed. 

6.  Differences  have  been  observed  in  the  crystalline  form  of  oxy- 
haemoglobin  and  haemoglobin  from  the  blood  of  the  same  species  in 
certain  cases. 

The  follo\\dng  bodies  may  be  derived  from  haemoglobin,  and  each 
possesses  a  specific  spectrum  which  serves  as  an  aid  in  its  detection 
and  identification:  Oxyhaemoglobin,  methaemoglobin,  carbon-monoxide 
haemoglobin,  nitric-oxide  haemoglobin,  haemochromogen,  haematin, 
acid-hasmatin,  alkah-hsematin  and  haematoporphyrin  (see  Absorption 
Spectra,  Plates  I  and  II). 

The  white  corpuscles  (or  leucocytes)  of  human  blood  differ  from 
the  red  corpuscles  (or  erythrocytes)  in  many  particulars,  such  as  being 
somewhat  larger  in  size,  in  containing  at  least  a  single  nucleus  and  in 
possessing  amoeboid  movement  (see  Plate  IV,  opposite  page  196). 
They  are  typical  animal  cells  and  therefore  contain  the  following  bodies 
which  are  customarily  present  in  such  cells:  Proteins,  fats,  glycogen, 
purine  bodies,  enzymes,  phosphatides,  lecithin,  cholesterol,  inorganic  salts 
and  water.  Compound  proteins  make  up  the  chief  part  of  the  protein 
quota  of  leucocytes,  the  nucleo-proteins  predominating.  Of  the  enzymes 
present  the  proteolytic  are  the  most  important.  It  is  claimed^  that 
there  are  two  proteolytic  enzymes  in  leucocytes,  one  active  in  alkaline 
solution  and  present  in  the  polynuclear  cells  ^  and  the  other  active  in  acid 
medium  and  present  in  mononuclear  cells.  It  is  claimed  that  the  granu- 
lar leucocytes  originate  in  the  bone  marrow,  whereas  the  non-granular 
leucocytes  (lymphocytes)  have  a  lymphatic  origin  (lymph  glands  or 
lymphoid  tissue);  this  matter  of  origin  is  uncertain.  The  normal  number 
of  leucocytes  in  human  blood  varies  between  5000  and  10,000  per  cubic 
milhmeter.  The  ratio  between  the  leucocytes  and  erythrocytes  is  about 
1 : 3 50-500.  A  leucocytosis  is  said  to  exist  when  the  number  of  leucocyte  s 
is  increased  for  any  reason.  Leucocytoses  may  be  divided  into  two 
general  classes,  the  physiological  and  the  pathological.  Under  the 
physiological  form  would  be  classed  those  leucocytoses  accompanying 
pregnancy,  parturition  and  digestion,  as  well  as  those  due  to  mechanical 
and  thermal  influences.  The  leucocytoses  spoken  of  as  pathological  are 
the  inflammatory,  infectious,  post-haemorrhagic,  toxic  and  experimental 
forms  as  well  as  the  type  of  leucocytosis  which  accompanies  maHgnant 
disease. 

*  Opie:    Jour,  of  Experimental  Med.,  8;  Opie  and  Barker:  Ibid.,  g. 

^For  discussion  of  different  types  of  leucocytes,  see  "Da  Costa's  Clinical  Hematology" 
or  some  similar  volume. 


BLOOD  AND    LYMPH.  203 

The  blood  plates  (platelets  or  plaques)  arc  round  or  oval,  colorless 
discs  which  possess  a  diameter  about  one-third  as  great  as  that  of  the 
erythrocytes.  Upon  treatment  with  certain  reagents,  c.  3^.,  artificial 
gastric  juice,  they  may  be  separated  into  a  homogeneous,  non-refractive 
portion  and  a  granular,  refractive  portion.  The  blood  plates  are  probably 
associated  in  some  way  with  the  coagulation  of  the  blood.  This  relation- 
ship is  not  well  understood  at  present. 

The  haemoconein  or  so-called  ''blood  dust"  is  made  up  of  round 
granules  which  usuallv  have  a  diameter  somewhat  less  than  one  micron. 
The  serum  of  normal  as  well  as  of  pathological  blood  contains  these 
granules.  They  were  first  described  by  Miiller  to  whom  they  appeared 
as  highly  refractile  granules  possessed  of  Brownian  movement.  The 
"blood  dust"  is  apparently  not  concerned  with  the  coagulation  of  the 
blood.  The  granules  are  insoluble  in  alcohol,  ether  and  acetic  acid 
and  are  not  blackened  by  osmic  acid.  According  to  Miiller,  the  gran- 
ules making  up  the  so-called  "blood  dust"  constitute  a  new  organized 
constituent  of  the  blood,  whereas  other  investigators  believe  them  to  be 
merely  free  granules  from  certain  of  the  forms  of  leucocytes.  In  common 
with  blood  plates  the  "blood  dust"  possesses  no  clinical  significance. 

The  processes  involved  in  the  coagulation  of  the  blood  are  not  fully 
understood.  Several  theories  have  been  advanced  and  each  has  its 
adherents.  The  theory  which  appears  to  be  fully  as  firmly  founded 
upon  experimental  evidence  as  any  is  the  following:  Blood  contains 
a  zymogen  called  prothrombin  which  combines  with  the  calcium  salts 
present  to  form  an  enzyme  known  as  thrombin  or  fibrin-ferment.  When 
freshly  drawn  blood  comes  in  contact  with  the  air  the  fibrin-ferment  at 
once  acts  upon  the  fibrinogen  present  and  gives  rise  to  the  formation 
of  fibrin.  This  fibrin  forms  in  shreds  throughout  the  blood  mass  and, 
holding  the  form  elements  of  the  blood  within  its  meshes,  serves  to 
produce  the  typical  blood  clot.  The  fibrin  shreds  gradually  contract, 
the  whole  clot  assumes  a  jelly-like  appearance  and  the  yellowish  serum 
exudes.  If,  immediately  upon  the  withdrawal  of  blood  from  the  body, 
the  fluid  be  rapidly  stirred  or  thoroughly  "whipped"  with  a  bundle 
of  coarse  strings,  twigs  or  a  specially  constructed  beater,  the  fibrin  shreds 
will  not  form  in  a  network  throughout  the  blood  mass  but  instead  will 
cling  to  the  device  used  in  beating.  In  this  way  the  fibrin  may  be  re- 
moved and  the  remaining  fluid  is  termed  dejibrinated  blood.  The  above 
theory  of  the  coagulation  of  the  blood  may  be  stated  briefly  as  follows: 

I.  Prothrombin -f  Calcium  Salts  =  Thrombin  (or  Fibrin-ferment). 

II.  Thrombin  (or  Fibrin-ferment) -f  Fibrinogen  =Fibrin. 

HowelP  has  very  recently  suggested  an  ingenious  modification  of  the 

'  Howell:    .American  Journal  of  Physiology,  29,  187,  191 1. 


204  PHYSIOLOGICAL   CHEMISTRY, 

above  theory.  He  says  "In  the  circulating  blood  we  find  as  constant 
constituents,  fibrinogen,  prothrombin,  calcium  salts  and  antithrombin. 
The  last-named  substance  holds  the  prothrombin  in  combination  and 
thus  prevents  its  conversion  or  activation  to  thrombin.  When  the  blood 
is  shed,  the  disintegration  of  the  corpuscles  (platelets)  furnishes  material 
(thromboplastin)  which  combines  with  the  antithrombin  and  liberates 
the  prothrombin;  the  latter  is  then  activated  by  the  calcium  and  acts  on 
the  fibrinogen.  According  to  this  view  the  actual  process  of  coagulation 
involves  only  three  factors,  fibrinogen,  prothrombin  and  calcium.  These 
three  factors  exist  normally  in  the  circulating  blood,  but  are  prevented 
from  reacting  by  the  presence  of  antithrombin." 

Among  the  medico-legal  tests  for  blood  are  the  following:  (i) 
Microscopical  identification  of  the  erythrocytes,  (2)  spectroscopic 
identification  of  blood  solutions,  (3)  the  guaiac  test,  (4)  the  benzidine 
reaction,  (5)  preparation  of  haemin  crystals.  Of  these  five  tests  the 
two  last  named  are  generally  considered  to  be  the  most  satisfactory. 
They  give  equally  reliable  results  with  fresh  blood  and  with  blood  from 
clots  or  stains  of  long  standing,  provided  the  latter  have  not  been  exposed 
to  a  high  temperature,  or  to  the  rays  of  the  sun  for  a  long  period.  The 
technic  of  the  tests  is  simple  and  the  formation  of  the  dark  brown 
or  chocolate  colored  crystals  of  haemin  or  the  production  of  the  green  or 
blue  color  with  benzidine  is  indisputable  proof  of  the  presence  of  blood 
in  the  fluid,  clot  or  stain  examined.  The  weak  point  of  the  tests,  medico 
legally,  lies  in  the  fact  that  they  do  not  differentiate  between  human  blood 
and  that  of  certain  other  species  of  animal. 

The  guaiac  test  (see  page  209),  although  generally  considered  less 
accurate  than  the  haemin  test,  is  really  a  more  delicate  test  than  the  haemin 
test  if  properly  performed.  One  of  the  most  common  mistakes  in  the 
manipulation  of  this  test  is  the  use  of  a  guaiac  solution  which  is  too  con- 
centrated and  which,  when  brought  into  contact  with  the  aqueous  blood 
solution,  causes  the  separation  of  a  voluminous  precipitate  of  a  resinous 
material  which  may  obscure  the  blue  coloration:  this  is  particularly  true 
of  the  test  when  used  for  the  examination  of  blood  stains.  A  solution  of 
guaiac  made  by  dissolving  i  gram  of  the  resin  in  60  c.c.  of  95  per  cent 
alcohol  is  very  satisfactory  for  general  use.  The  test  is  frequently  objected 
to  upon  the  ground  that  various  other  substances,  e.  g.,  milk,  pus,  saliva, 
etc.,  respond  to  the  test  and  that  it  cannot  therefore  be  considered  a 
specific  test  for  blood  and  is  of  value  only  in  a  negative  sense.  We  have 
demonstrated  to  our  own  satisfaction,  however,  that  milk  many  times 
gives  the  blue  color  upon  the  addition  of  an  alcoholic  solution  of 
guaiac  resin  without  the  addition  of  hydrogen  peroxide  or  old  turpentine. 
Buckmaster  has  advocated  the  use  of  an  alcoholic  solution  of  guaia- 


BLOOD   AND    LYMPH.  205 

conic  acid  instead  of  an  alcoholic  solution  of  guaiac  resin.  He  claims 
that  he  was  able  to  produce  the  blue  color  ujK)n  the  addition  of  the 
guaiaconic  acid  to  milk  only  when  the  sample  of  milk  tested  was 
brought  from  the  country  in  sterile  bottles,  and  further,  that  no  sample  of 
London  milk  which  he  examined  responded  to  the  test.  In  the  applica- 
tion of  the  guaiac  test  to  the  detection  of  blood,  he  states  that  he  was  able 
"to  detect  laked  blood  when  present  in  the  ratio  i  :  5,000,000  and  unlaked 
blood  when  present  in  the  ratio  i  :  1,000.000.  This  author  considers  the 
guaiac  test  to  be  far  more  trustworthy  than  is  generally  believed. 

Up  to  within  recent  times  it  has  been  impossible  to  make  an  absolute 
difTercntiation  of  human  blood.  Recently,  however,  the  so-called 
"biological"  blood  test  has  made  such  a  differentiation  possible.  This 
test,  known  as  the  Bordet  reaction,  is  founded  upon  the  fact  that  the 
blood  scrum  of  an  animal  into  which  has  been  injected  the  blood  of 
another  animal  of  different  species  develops  the  property  of  agglutinating 
and  dissolving  erythrocytes  similar  to  those  injected,  but  exerts  this  influence 
■upon  the  blood  from  no  other  species.  The  antiserum  used  in  this  test  is 
prepared  by  injecting  rabbits  with  5-10  c.c.  of  human  defibrinated  blood, 
at  intervals  of  about  four  days  until  a  total  of  between  50  and  80  c.c.  has 
been  injected.  After  a  lapse  of  one  or  two  weeks  the  animal  is  bled,  the 
serum  collected,  placed  in  sterile  tubes  and  preserved  for  use  as  needed. 
In  examining  any  specific  solution  for  human  blood  it  is  simply  necefsary 
to  combine  the  antiserum  and  the  solution  under  examination  in  the 
proportion  of  i  :  100  and  place  the  mixture  at  37°  C,  If  human  blood  is 
present  in  the  solution  a  turbidity  will  be  noted  and  this  will  change 
within  three  hours  to  a  distinctly  flocculent  precipitate.  This  antiserum 
will  react  thus  with  no  other  known  substance. 

Lymph  may  be  considered  as  the  "middle  man"  in  the  transactions 
between  blood  and  tissues.  It  is  the  medium  by  which  the  nutritive 
material  and  oxygen  transported  by  the  blood  for  the  tissues  is  brought  into 
intimate  contact  with  those  tissues  and  thus  utilized.  In  the  further  ful- 
fillment of  its  function,  the  lymph  bears  from  the  tissues  \vater,  salts  and 
the  products  of  the  activity  and  catabolism  of  the  tissues  and  passes  these 
into  the  blood.  Lymph,  therefore,  exercises  the  function  of  a  "go-between" 
for  blood  and  tissues.  It  bathes  every  active  tissue  of  the  animal  body, 
and  is  believed  to  have  its  origin  partly  in  the  blood  and  partly  in  the 
tissues. 

In  chemical  characteristics,  lymph  resembles  blood  plasma.  In  fact, 
it  has  been  termed  "blood  without  its  red  corpuscles."  Lymph  from  the 
thoracic  duct  of  a  fasting  animal  or  from  a  large  lymphatic  vessel  of  a  well- 
nourished  animal  is  of  a  variable  color  (colorless,  yellowish  or  slightly 
reddish)  and  alkaline  in  reaction  to  litmus.     It  contains  fibrinogen,  fibrin 


2o6  PHYSIOLOGICAL   CHEMISTRY. 

ferment  and  leucocytes  and  coagulates  slowly,  the  clot  being  less  firm  and 
bulky  than  the  blood  clot.  Serum  albumin  and  serum  globulin  are  both 
present  in  lymph,  the  albumin  predominating  in  a  ratio  of  about  3  or  4  :  i. 
The  principle  inorganic  salts  are  sodium  salts  (chloride  and  carbonate), 
whereas  the  phosphates  of  potassium,  calcium,  magnesium  and  iron  are 
present  in  smaller  amount. 

Substances  which  stimulate  the  flow  of  lymph  are  termed  lympha- 
gogues.  Such  substances,  as  sugar,  urea,  certain  salts  (especially  sodium 
chloride)  peptone,  egg  albumin,  extracts  of  dogs'  liver  and  intestine,  crab 
muscles  and  blood  leeches  are  included  in  this  class. 

In  a  fasting  animal,  the  lymph  coming  from  the  intestine  is  a  clear, 
transparent  fluid  possessing  the  characteristics  already  outlined.  After 
a  meal  containing  fat  has  been  ingested,  this  intestinal  lymph  is  white 
or  "milky."  This  is  termed  chyle  and  is  essentially  lymph  possessing  an 
abnormally  high  (5-15  per  cent)  content  of  emulsified  fat.  This  chyle 
is  absorbed  by  the  lacteals  of  the  intestine  and  transported  to  the  lower 
portion  of  the  thoracic  duct.  Apart  from  the  fat  value,  the  composition  of 
lymph  and  chyle  are  similar. 

Experiments  ox  Blood. 
I.  Defibrinated  Ox-blood. 

1.  Reaction. — Moisten  red  and  blue  litmus  papers  with  10  per  cent 
sodium  chloride  solution  and  test  the  reaction  of  the  defibrinated  blood. 
Test  by  congo-red  paper  also. 

2.  Microscopical  Examination. — Examine  a  drop  of  defibrinated 
blood  under  the  microscope.  Compare  the  objects  you  observ^e  ^ith 
Plate  IV,  opposite  page  196.  Repeat  the  test  with  a  drop  of  your  own 
blood. 

3.  Specific  Gravity. — Determine  the  specific  gra\'ity  of  defibrinated 
blood  by  means  of  an  ordinary  specific  gravity  spindle.  Compare  this 
result  with  the  specific  gravity  as  determined  by  Hammerschlag's  method 
in  the  next  experiment. 

4.  Specific  Gravity  by  Hammerschlag's  Method. — Fill  an  ordinary 
urinometer  cylinder  about  one-half  full  of  a  mixture  of  chloroform  and 
benzene,  having  a  specific  gravity  of  approximately  1.050.  Into  this 
mixture  allow  a  drop  of  the  blood  under  examination  to  fall  from  a  pipette  or 
directly  from  the  finger  in  case  fresh  blood  is  being  examined.  Care  must 
be  taken  not  to  use  too  large  a  drop  of  blood  and  to  keep  the  drop  from 
coming  in  contact  with  the  walls  of  the  cylinder.  If  the  blood  drop  sinks 
to  the  bottom  of  the  vessel,  thus  showing  it  to  be  of  higher  specific  gravity 
than  the  surrounding  fluid,  add  chloroform  until  the  blood  drop  remains 


BLOOD   AND    LYMPH.  207 

suspended  in  the  mixture.  Stir  carefully  with  a  glass  rod  after  adding  the 
chloroform.  If  the  blood  drop  rises  to  the  surface  upon  being  introduced 
into  the  mixture,  thus  showing  it  to  be  of  lower  specific  gravity  than  the 
surrounding  fluid,  add  benzene  until  the  blood  drop  remains  suspended  in 
the  mixture.  Stir  with  a  glass  rod  after  the  benzene  is  added.  After  the 
blood  drop  has  been  brought  to  a  suspended  position  in  the  mixture  by 
means  of  one  or  more  additions  of  chloroform  and  benzene  this  final 
mixture  should  Be  filtered  through  muslin  and  its  specific  gravity  accurately 
determined.     What  is  the  specific  graWty  of  the  blood  under  examination  ? 

5.  Tests  for  Various  Constituents. — Place  lo  c.c.  of  defibrinated 
blood  in  an  evaporating  dish,  dilute  with  loo  c.c.  of  water  and  heat  to 
boiling.  Is  there  any  coagulation,  and  if  so  what  bodies  form  the  coag- 
ulum?  At  the  boiling-point  acidulate  slightly  with  dilute  acetic  acid. 
Filter.  The  filtrate  should  be  clear  and  the  coagulum  dark  brown.  Re- 
serve this  coagulum.  What  body  gives  the  coagulum  this  color  ?  Evap- 
orate the  filtrate  to  about  25  c.c,  filtering  off  any  precipitate  which  may 
form  in  the  process.     Make  the  following  tests  upon  the  filtrate: 

(a)  Fehlings  Test. — Test  for  sugar  according  to  directions  given  on 
page  32. 

{h)  Chlorides. — To  a  small  amount  of  the  filtrate  in  a  test-tube  add 
a  few  drops  of  nitric  acid  and  a  little  silver  nitrate.  In  the  presence 
of  chloride,  a  white  precipitate  of  silver  chloride  will  form. 

(c)  Phosphates. — Test  for  phosphates  by  nitric  acid  and  molybdic 
solution  according  to  directions  given  on  page  64. 

{d)  Proteose  and  Peptone. — Test  a  small  amount  of  the  solution  for 
proteose  and  peptone  by  saturating  with  ammonium  sulphate  according 
to  directions  given  on  page  120. 

{e)  Crystallizatmi  of  Sodium  Chloride. — Place  the  remainder  of  the 
filtrate  in  a  watch  glass  and  evaporate  it  on  a  water-bath.  Examine 
the  crystals  under  the  microscope  and  compare  them  with  those  in 
Fig.  61,  page  213. 

6.  Test  for  Iron.- — Incinerate  a  small  portion  of  the  coagulum  from 
the  last  experiment  (5)  in  a  porcelain  crucible.  Cool,  dissolve  the  residue 
in  dilute  hydrochloric  acid  and  test  for  iron  by  potassium  ferrocyanide 
or  ammonium  thiocyanate.  Which  of  the  constituents  of  the  blood 
contains  the  iron  ? 

7.  Haemolysis  ("Laky  Blood)." — Note  the  opacity  of  ordinary  defibri- 
nated blood.  Place  a  few  cubic  centimeters  of  this  blood  in  a  test-tube 
and  add  water,  a  little  at  a  time,  until  the  blood  is  rendered  transparent. 
Hcemolysis  has  taken  place.  How  does  the  water  act  in  causing  this 
transparency?  Examine  a  drop  of  haemolyzed  blood  under  the  micro- 
scope.    How   does   its   microscopical   appearance   differ   from   that   of 


2o8  PHYSIOLOGICAL   CHEMISTRY. 

unaltered   blood?     What   other   agents   may   be   used   to   bring   about 
haemolysis  ? 

8.  Osmotic  Pressure. — ^Place  a  few  cubic  centimeters  of  blood  in 
each  of  three  test-tubes.  Haemolyze  the  blood  in  the  first  tube  according 
to  directions  given  in  the  last  experiment  (7) :  add  an  equal  volume  of 
isotonic  (0.9  per  cent)  sodium  chloride  to  the  blood  in  the  second  tbue, 
and  an  equal  volume  of  10  per  cent  sodium  chloride  to  the  blood  in  the 
third  tube.  Mix  thoroughly  by  shaking  and  after  a  few  moments  exam- 
ine a  drop  from  each  of  the  three  tubes  under  the  microscope  (see  Figs. 
58  and  120,  below  and  p.  377  ).  What  do  you  find  and  what  is  your 
explanation  from  the  standpoint  of  osmotic  pressure  ? 


Fig.  58. — Efpect  of  Water  on  Erythrocytes. 

9.  Haemagglutination. — The  common  garden  bean,  such  as  the 
Scarlet  Runner/  contains  a  protein  substance  which  exhibits  the  inter- 
esting property  of  causing  a  clumping  or  agglutination  of  red  blood 
corpuscles.^ 

Dilute  defebrinated  blood^  ten  times  with  physiological  sodium 
chloride  solution  (0.9  per  cent)  and  place  i  c.c.  in  each  of  three  small  test- 
tubes. 

Grind  three  beans  in  a  coffee  mill,  or  with  mortar  and  pestle  to  a  fine 
meal  and  extract  for  a  few  minutes  with  0.9  per  cent  sodium  chloride 
solution.     Filter  and  add  0.05  c.c.    (about  2-3  drops)  of   the  filtered 

'  The  Scarlet  Runner  is  a  familar  variety  purchasable  in  every  seed  store.  Ricin  a  protein 
constituent  of  the  castor  bean  also  possesses  pronounced  agglutinating  properties.  Because 
of  its  poisonous  nature  it  is,  however,  not  suitable  for  use  in  class  experiments. 

^Mendel:  Archivio  di  fisiologia,  7,  168,  1909;  Schneider:  Journal  Biol.  Chem.,  11,  47, 
1912. 

'  Rabbit's  blood  is  especially  desirable  (Mendel:  Loc.  cit.)  and  may  be  obtained  for  the 
purpose  by  bleeding  from  a  small  cut  on  the  animal's  ear  and  defibrinating. 


BLOOD  AND    LYMPH.  209 

extract  to  the  first  of  the  blood  tubes;  o.oi  c.c.  to  the  second;  and  0.05 
of  0.9  per  cent  sodium  chloride  solution  to  the  third. 

Invert  each  tube  to  mix  the  contents  thoroughly,  and  note  the  rapid 
agglutination,  and  precipitation  of  the  blood  corpuscles  in  the  first 
tube,  a  less  rapid  agglutination  in  the  second,  while  the  third  or 
control  tube  remains  unaltered.  In  one-half  hour  the  corpuscles  in 
the  first  tube  ^often  are  packed  solid  and  one  is  able  to  pour  off  perfectly 
clear  serum. 

If  the  remainder  of  the  bean  extract  is  boiled  for  a  few  minutes,  the 
coagulum  filtered  out  and  0.05  c.c.  of  the  filtrate  added  to  the  control 
tube,  still  no  agglutination  occurs,  indicating  that  the  hasmagglutinin 
has  been  destroyed  or  removed  by  the  boiling. 

10.  Diffusion  of  Haemoglobin.— Prepare  some  hcBtnolyzed  ("laky") 
blood,  thus  liberating  the  haemoglobin  from  the  erythrocytes.  Test  the 
diffusion  of  the  haemoglobin  by  preparing  a  dialyzer  like  one  of  the  models 
shown  in  Fig.  2,  page  30.  How  does  haemoglobin  differ  from  other  well- 
known  crystallizable  bodies  ? 

11.  Guaiac  Test. — To  5  c.c.  of  water  in  a  test-tube  add  two  drops 
of  blood.  By  means  of  a  pipette  drop  an  alcoholic  solution  of  guaiac 
(strength  about  1:60)^  into  the  resulting  mixture  until  a  turbidity  is 
observed  and  add  old  turpentine  or  hydrogen  peroxide,  drop  by  drop, 
until  a  blue  color  is  obtained.  Do  any  other  substances  respond  in  a 
similar  manner  to  this  test  ?  Is  a  positive  guaiac  test  a  sure  indication  of 
the  presence  of  blood  ? 

12.  Schiunm's  Modification  of  the  Guaiac  Test. — To  about  5  c.c. 
of  the  solution  under  examination^  in  a  test-tube  add  about  ten  drops 
of  freshly  prepared  alcoholic  solution  of  guaiac.  Agitate  the  tube  gently, 
add  about  20  drops  of  old  turpentine,  subject  the  tube  to  a  thorough 
shaking  and  permit  it  to  stand  for  about  2-3  minutes.  A  blue  color 
indicates  the  presence  of  blood  in  the  solution  under  examination.  In 
case  there  is  insufficient  blood  to  yield  a  blue  color  under  these 
conditions,  a  few  c.c.  of  alcohol  should  be  added  and  the  tube  gently 
shaken,  where-upon  a  blue  coloration  will  appear  in  the  upper  alcohol- 
turpentine  layer. 

A  control  test  should  always  be  made,  using  water  in  place  of  the 
solution  under  examination.  In  the  detection  of  very  minute  traces  of 
blood  only  3-5  drops  of  the  guaiac  solution  should  be  employed. 

13.  Adler's  Benzidine  Reaction. — This  is  one  of  the  most  delicate 
of  the  reactions  for  the  detection  of  blood.     Different  benzidine  prepara- 

'  Buckmaster  advises  the  use  of  an  alcoholic  solution  of  guaiaconic  acid  instead  of  an 
alcoholic  solution  of  guaiac  resin. 

-  Alkaline  solutions  should  be  made  slightly  acid  with  acetic  acid,  as  the  blue  end-reaction 
is  very  sensitive  to  alkali. 

14 


210  PHYSIOLOGICAL   CHEMISTRY. 

tions  vary  greatly  in  their  sensitiveness,  however.  Inasmuch  as  benzidine 
solutions  change  readily  upon  contact  with  light  it  is  essential  that  they 
be  kept  in  a  dark  place.  The  test  is  performed  as  follows :  To  a  saturated 
solution  of  benzidine  in  alcohol  or  glacial  acetic  acid  add  an  equal  volume 
of  3  per  cent  hydrogen  peroxide  and  one  c.c.  of  the  solution  under  exami- 
nation. If  the  mixture  is  not  already  acid  render  it  so  with  acetic  acid, 
and  note  the  appearance  of  a  green  or  blue  color.  A  control  test  should 
be  made  substituting  water  for  the  solution  under  examination.  The 
sensitiveness  of  the  benzidine  reaction  is  greater  when  applied  to  aqueous 
solutions  than  when  applied  to  the  urine.  According  to  Ascarelli^  the 
benzidine  reaction  serves  to  detect  blood  when  present  in  a  dilution  of 
I  :  300,000.  Walter^  has  also  recently  shown  the  test  to  be  very  delicate 
and  claims  it  to  be  more  satisfactory  than  the  guaiac  test. 

14.  Haemin  Test. — (a)  Teichmann''s  Method. — ^Place  a  very  small 
drop  of  blood  on  a  microscopic  slide,  add  a  minute  grain  of  sodium 
chloride^  and  carefully  evaporate  to  dryness  over  a  low  flame.  Put  a 
cover  glass  in  place,  run  underneath  it  a  drop  of  glacial  acetic  acid  and 
•warm  gently  until  the  formation  of  gas  bubbles  is  noted.  Add  another 
drop  of  glacial  acetic  acid,  cool  the  preparation,  examine  under  the 
microscope  and  compare  the  crystals  with  those  shown  in  Figs.  59  and  60, 
page  211.  The  haemin  crystals  result  from  the  decomposition  of  the 
haemoglobin  of  the  blood.  What  are  the  steps  involved  in  this  process  ? 
The  haemin  crystals  are  also  called  Teichmann's  crystals.  Is  this  an 
absolute  test  for  blood?  Is  it  possible  to  differentiate  between  human 
blood  and  the  blood  of  other  species  by  means  of  the  haemin  test  ?    . 

{h)  Atkinson  and  Kendall's  Method. — Introduce  a  small  amount  of 
the  solution  under  examination  into  a  tube  closed  at  one  end,  add  sodium 
chloride  and  glacial  acetic  acid  as  in  Teichmann's  method,*  fuse  or 
tightly  plug  the  open  end  of  the  tube  and  heat  for  fifteen  minutes  in  a 
boiling  water-bath.^  Remove  the  tube  and  permit  it  to  cool  to  room 
temperature  spontaneously.  When  the  tube  has  cooled,  break  it  open, 
transfer  the  contents  to  a  watch  glass  or  small  evaporating  dish  and 
concentrate  on  a  water-bath  until  the  volume  of  the  fluid  in  the  watch 
glass  or  dish  has  been  reduced  to  a  few  drops.  Transfer  a  drop  of  this 
fluid  to  a  slide,  cover  with  a  cover  slip,  allow  the  slide  to  stand  for  a  few 
minutes  and  examine  it  under  a  microscope.  Compare  the  crystals 
with  those  shown  in  Figs.  59  and  60,  page  211.  In  case  crystals  of  sodium 
chloride  (see  Fig.  61,  page  213)  obstruct  the  view  of  the  haemin  crystals, 

*  Ascarelli:    II  policlin  sez.  prat.,  igog. 

^  Walter:    Deul.  med.  Woch.,  36   p.  3og. 

^  Buckmaster  considers  the  use  of  potassium  chloride  preferable. 

*  Care  should  be  taken  not  to  add  too  great  an  excess  of  these  reagents. 
'  This  process  insues  constancy  of  temperature  and  strength  of  reagents. 


BLOOD   AND    LYMPH. 


211 


>f 


^   W^<^    WrA  '<>^ 

Fig.  59. — H^MiN  Crystals  from  Human  Blood. 
Reproduced  from  a  micro-photograph  furnished  by  Prof.  E.  T.  Reichert,  of  the  University  of 

Pennsylvania. 


^"^Z 


Fig.  60. — H.EMIN  Crystals  from  Sheep  Blood. 
Reproduced  from  a  micro-photograph  furnished  by  Prof.  E.  T.  Reichert,  of  the  University  of 

Pennsylvania. 


212  PHYSIOLOGICAL   CHEMISTRY. 

dissolve  the  sodium  chloride  crystals  by  running  a  drop  of  water  under 
the  cover  slip. 

(c)  V.  Zeynek  and  Nencki^s  Method. — To  lo  c.c.  of  defibrinated 
blood  add  acetone  until  no  more  precipitate  forms.  Filter  off  the  pre- 
cipitated protein  and  extract  it  with  lo  c.c.  of  acetone  made  acid  with 
2-3  drops  of  hydrochloric  acid.  Place  a  drop  of  the  resulting  colored 
extract  on  a  slide,  immediately  place  a  cover  glass  in  position  and  examine 
under  the  microscope.  Upon  the  evaporation  of  the  acetone,  crystals 
of  haemin  will  form.  Larger  crystals  may  be  obtained  by  evaporating 
the  acetone  extract  about  one-half,  transferring  it  to  a  stoppered  vessel 
and  allowing  it  to  remain  overnight. 

(d)  Schalfijew^s  Method. — ^Place  20  c.c.  of  glacial  acetic  acid  in  a 
small  beaker  and  heat  to  80°  C.  Add  5  c.c.  of  strained  defibrinated  blood, 
again  bring  the  temperature  to  80°  C,  remove  the  flame  and  allow  the 
mixture  to  cool.  Examine  the  crystals  under  the  microscope  and  compare 
them  with  those  reproduced  in  Figs.  59  and  60,  page  211. 

15.  Catalytic  Action. — To  about  10  drops  of  blood  in  a  test-tube 
add  twice  the  volume  of  hydrogen  peroxide,  without  shaking.  The 
mixture  foams.     What  is  the  cause  of  this  phenomenon? 

16.  Preparation  of  Haematin. — ^Place  100  c.c.  of  hcemolyzed  {laked) 
blood  in  a  beaker  and  add  95  per  cent  alcohol  until  precipitation  ceases. 
What  bodies  are  precipitated?  Transfer  the  precipitate  to  a  flask  and 
boil  with  95  per  cent  alcohol  previously  acidulated  with  sulphuric  acid. 
Through  the  action  of  the  acid  the  haemoglobin  is  split  into  haematin  and  a 
protein  body  called  globin.  Later  the  "sulphuric  acid  ester  of  haematin" 
is  formed,  which  is  soluble  in  the  alcohol.  Continue  heating  until  the 
precipitate  is  no  longer  colored,  then  filter.  Partly  saturate  the  filtrate 
with  sodium  chloride  and  warm.  In  this  process  the  "hydrochloric  acid 
ester  of  haematin"  is  formed.  Filter  and  dissolve  on  the  filter  paper  by 
sodium  carbonate.  Save  this  alkaline  solution  of  haematin  and  make  a 
spectroscopic  examination  later  after  becoming  familiar  with  the  use  of 
the  spectroscope.  How  does  the  spectrum  of  oxy haemoglobin  differ  from 
that  of  the  derived  alkali  hcematin? 

17.  Variation  in  Size  of  Erythrocytes. — Prepare  two  small  funnels 
with  filter  papers  such  as  are  used  in  quantitative  analysis.  Moisten  each 
paper  with  physiological  (isotonic)  salt  solution.  Into  one  funnel  intro- 
duce a  small  amount  of  defibrinated  ox  blood  and  into  the  other  funnel 
allow  blood  to  drop  directly  from  a  decapitated  frog.  Note  that  the 
filtrate  from  the  ox  blood  is  colored  whereas  that  from  the  frog  blood  is 
colorless.  What  deduction  do  you  make  regarding  the  relative  size  of  the 
erythrocytes  in  ox  and  frog  blood  ?     Does  either  filtrate  clot  ?     Why  ? 


BLOOD   AND    LYMPH. 


II.  Blood  Serum. 


213 


1.  Coagulation  Temperature. — ^Placc  5  c.c.  of  undiluted  serum  in  a 
test-tube  and  determine  its  temperature  of  coagulation  according  to  the 
method  described  on  page  106.  Note  the  temperature  at  which  a  cloudi- 
ness occurs  as  well  as  the  temperature  at  which  coagulation  is  complete. 

2.  Precipitation  by  Alcohol. — To  5  c.c.  of  serum  in  a  test-tube  add 
twice  the  amount  of  95  per  cent  alcohol  and  thoroughly  mix  by  shaking. 
What  is  this  precipitate  ?  Make  a  confirmatory  test.  Test  the  alcoholic 
filtrate  for  protein.     Explain  the  result. 

3.  Proteins  of  Blood  Serum.— Place  about  10  c.c.  of  serum  in  a 
small  evaporating  dish,  dilute  with  5  c.c.  of  water  and  heat  to  boiling. 
At  the  boiling-point  acidify  slightly  with  dilute  acetic  acid.  Of  what 
does  this  coagulum  consist  ?  Filter  off  the  coagulum  (reserve  the  filtrate) 
and  test  it  as  follows: 

(a)  Millori's  Reactian. — Make  the  test  according  to  directions  given 
on  page  97, 

{h)  Hopkins-Cole  Reaction. — Make  the  test  according  to  directions 
given  on  page  98. 


tJ^  /'^. 


i^  J  ' 


Fig.  61. — Sodium  Chloride. 


4.  Sugar  in  Serum. — Test  a  little  of  the  filtrate  from  Experiment  3 
by  Fehling's  test.     What  do  you  conclude  ? 

5.  Detection  of  Sodium  Chloride. — {a)  Test  a  little  of  the  filtrate 
from  Experiment  3  for  chlorides,  by  the  use  of  nitric  acid  and  silver 
nitrate,  (b)  Evaporate  5  c.c.  of  the  filtrate  from  Experiment  3  in  a  watch 
glass  on  a  water-bath.  Examine  the  crystals  and  compare  them  with  those 
reproduced  in  Fig.  61,  above. 

*  For  directions  as  to  preparation  of  serum,  see  Appendix. 


214  PHYSIOLOGICAL    CHEMISTRY. 

6.  Separation  of  Serum  Globulin  and  Serum  Albtmiin. — Place 
lo  c.c.  of  blood  serum  in  a  small  beaker  and  saturate  with  magnesium 
sulphate.  What  is  this  precipitate?  Filter  it  off  and  acidify  the  filtrate 
slightly  with  acetic  acid.  What  is  this  second  precipitate?  Filter  this 
precipitate  off  and  test  the  filtrate  by  the  biuret  test.  What  do  you  con- 
clude ? 

III.  Blood  Plasma. 

1.  Preparation  of  Oxalated  Plasma. — Allow  arterial  blood  to  run 
into  an  equal  volume  of  0.2  per  cent  ammonium  oxalate  solution. 

2.  Preparation  of  Fibrinogen. — To  25  c.c.  of  oxalated  plasma  add 
an  equal  volume  of  saturated  sodium  chloride  solution.  Note  the  pre- 
cipitation of  fibrinogen.  Filter  off  the  precipitate  (reserve  the  filtrate) 
and  test  it  by  a  protein  color  test  (see  page  97). 

3.  Effect  of  Calcium  Salts. — Place  a  small  amount  of  oxalated 
plasma  in  a  test-tube  and  add  a  few  drops  of  a  2  per  cent  calcium  chloride 
solution.     What  occurs  ?     Explain  it. 

4.  Preparation  of  Salted  Plasma. — Allow  arterial  blood  to  run  into 
an  equal  volume  of  a  saturated  solution  of  sodium  sulphate  or  a  10  per 
cent  solution  of  sodium  chloride.  Keep  the  mixture  in  a  cool  place  for 
about  twenty-four  hours. 

5.  Effect  of  Dilution. — Place  a  few  drops  of  salted  plasma  in  a  test- 
tube  and  dilute  it  with  10-15  volumes  of  water.  What  do  you  observe? 
Explain  it. 

6.  Crystallization  of  Oxyhaemoglobin.  Reicheri's  Method. — Add 
to  5  c.c.  of  the  blood  of  the  dog,  horse,  guinea-pig,  or  rat,  before  or  after 
laking,  or  defibrinating,  from  i  to  5  per  cent  of  ammonium  oxalate  in 
substance.  Plcice  a  drop  of  this  oxalated  blood  on  a  slide  and  examine 
under  the  microscope.  The  crystals  of  oxyhsemoglobin  will  be  seen  to 
form  at  once  near  the  margin  of  the  drop,  and  in  a  few  minutes  the  entire 
drop  may  be  a  solid  mass  of  crystals.  Compare  the  crystals  with  those 
shown  in  Figs.  51  to  57,  pages  198  to  201. 

IV.  Fibrin. 

I.  Preparation  of  Fibrin. — Allow  blood  to  flow  directly  from  the 
animal  into  a  vessel  and  rapidly  whip  it  by  means  of  a  bundle  of  twigs, 
a  mass  of  strong  cords,  or  a  specially  constructed  beater.  If  a  pure  fibrin 
is  desired  it  is  not  best  to  attempt  to  manipulate  a  large  volume  of  blood 
at  one  time.  After  the  fibrin  has  been  collected  it  should  be  freed  from 
any  adhering  blood  clots  and  washed  in  water  to  remove  further  traces  of 
blood.  The  pure  product  should  be  very  light  in  color.  It  may  be  pre- 
served under  glycerol,  dilute  alcohol,  or  chloroform  water. 


BLOOD   AND    LYMPH.  215 

2.  Solubility.  Try  the  solubility  of  small  shreds  of  freshly  prepared 
li])rin  in  the  usual  solvents. 

3.  Millon's  Reaction. — Make  the  test  according  to  directions  given 
on  page  97. 

4.  Hopkins-Cole  Reaction. — Make  the  test  according  to  directions 
given  on  page  g8. 

5.  Biuret  Test. — Make  the  test  according  to  directions  given  on  page 

98. 

V.  Detection  of  Blood  in  Stains  on  Cloth,  Etc. 

1,  Identification  of  Corpuscles. — If  the  stain  under  examination  is 
on  cloth  a  i)ortion  should  be  extracted  with  a  few  drops  of  glycerol  or 
physiological  (0.9  per  cent)  sodium  chloride  solution.  A  drop  of  this 
solution  should  then  be  examined  under  the  microscope  to  determine  if 
corpuscles  arc  present. 

2.  Tests  on  Aqueous  Extract. — A  second  portion  of  the  stain  should 
be  extracted  with  a  small  amount  of  water  and  the  following  tests  made 
upon  the  aqueous  extract: 

{a)  HcBmochromogen. — Make  a  small  amount  of  the  extract  alkaline 
by  potassium  hydroxide  or  sodium  hydroxide,  and  heat  until  a  brownish- 
green  color  results.  Cool  and  add  a  few  drops  of  ammonium  sulphide  or 
Stokes'  reagent  (see  page  216)  and  make  a  spectroscopic  examination. 
Compare  the  spectrum  with  that  of  haemochromogen  (see  Absorption 
Spectra,  Plate  II). 

{h)  HcBmin  Test. — Make  this  test  upon  a  small  drop  of  the  aqueous 
extract  according  to  the  directions  given  on  page  210. 

(c)  Guaiac  Test. — Make  this  test  on  the  aqueous  extract  according  to 
the  directions  given  on  page  209.  The  guaiac  solution  may  also  be 
applied  directly  to  the  stain  without  previous  extraction  in  the  following 
manner:  Moisten  the  stain  with  water,  and  after  allowing  it  to  stand 
several  minutes,  add  an  alcoholic  solution  of  guaiac  (strength  about  1:60) 
and  a  little  hydrogen  peroxide  or  old  turpentine.  The  customary  blue 
color  will  be  observed  in  the  presence  of  blood. 

(d)  Benzidine  Reaction. — Make  this  test  according  to  directions  given 
on  p.  209. 

(e)  Acid  Hamatin. — If  the  stain  fails  to  dissolve  in  water  extract  with 
acid  alcohol  and  examine  the  spectrum  for  absorption  bands  of  acid 
haematin  (see  Absorption  Spectra,  Plate  II). 

*  VI.  Spectroscopic  Examination  of  Blood. 
(For  Absorption  Spectra  see  Plates  I.  and  II.) 
Either  the  a7igiilar-\ision  spectroscope  (Figs.  63  and  64,  page  217)  or 
the  direct-\\%\on  spectroscope  (Fig.  62,  page  216)  may  be  used  in  making 


2l6  PHYSIOLOGICAL   CHEMISTRY. 

the  spectroscopic  examination  of  the  blood.  For  a  complete  description 
of  these  instruments  the  student  is  referred  to  any  standard  text-book  of 
physics. 

I.  Oxyhaemoglobin. — Examine  dilute  (1:50)  defibrinated  blood 
spectroscopically.  Note  the  broad  absorption-band  between  D  and  E. 
Continue  the  dilution  until  this  single  broad  band  gives  place  to  two 
narrow  bands,  the  one  nearer  the  D  line  being  the  narrower.  These  are 
the  typical  absorption-bands  of  oxyhaemoglobin  obtained  from  dilute 
solutions  of  blood.  Now  dilute  the  blood  very  freely  and  note  that  the 
bands  gradually  become  more  narrow  and,  if  the  dilution  is  sufficiently 
great,  they  finally  entirely  disappear. 


Fig.  62." — Direct- VISION  Spectroscope. 

2.  Haemoglobin  (so-called  Reduced  Haemoglobin). — To  blood  which 
has  been  diluted  sufficiently  to  show  well-defined  oxyhaemoglobin  absorp- 
tion-bands add  a  small  amount  of  Stokes'  reagent.  ^  The  blood  immedi- 
ately changes  in  color  from  a  bright  red  to  violet-red.  The  oxyhaemo- 
globin has  been  reduced  through  the  action  of  Stokes'  reagent  and  haemo- 
globin (so-called  reduced  haemoglobin)  has  been  formed.  This  has  been 
brought  about  by  the  removal  of  some  of  the  loosely  combined  oxygen 
from  the  oxyhaemoglobin.  Examine  this  haemoglobin  spectroscopically. 
Note  that  in  place  of  the  two  absorption  bands  of  oxyhaemoglobin  we  now 
have  a  single  broad  band  lying  almost  entirely  between  D  and  E.  This 
is  the  typical  spectrum  of  haemoglobin.  If  the  solution  showing  this 
spectrum  be  shaken  in  the  air  for  a  few  moments  it  will  again  assume  the 
bright  red  color  of  oxyhaemoglobin  and  show  the  characteristic  spectrum 
of  that  pigment. 

3.  Carbon  Monoxide  Haemoglobin.— The  peparation  of  this  pig- 
ment may  be  easily  accomplished  by  passing  ordinary  illuminating  gas^ 
through  defibrinated  ox-blood.  Blood  thus  treated  assumes  a  brighter 
tint  (carmine)  than  that  imparted  by  oxyhaemoglobin.  In  very  dilute 
solution  oxyhaemoglobin  appears  yellowish-red  whereas  carbon  monoxide 
haemoglobin  under  the  same  conditions  appears  bluish-red.     Examine  the 

'  Stokes'  reagent  is  a  solution  containing  2  per  cent  ferrous  sulphate  and  3  per  cent  tartaric 
acid.  When  needed  for  use  a  small  amount  should  be  placed  in  a  test-tube  and  ammonium 
hydroxide  added  until  the  precipitate  which  forms  on  the  first  addition  of  the  hydroxide  has 
entirely  dissolved.     This  produces  ammonium  ferrolartrale  which  is  a  reducing  agent. 

*  The  so-called  water  gas  with  which  ordinary  illuminating  gas  is  diluted  contains  usually 
as  much  as  20  per  cent  of  carbon  monoxide  (CO). 


BLOOD  AND   LYMPH. 


217 


carbon  monoxide  haemoglobin  solution  spectroscopically.  Observe  that 
the  spectrum  of  this  body  resembles  the  spectrum  of  oxyhaemoglobin  in 
showing  two  absorption-bands  between  D  and  E.     The  bands  of  carbon 


Fig.  63. — Angular-vision  Spectroscope  Arranged  for  Absorption  Analysis. 

monoxide  haemoglobin,  however,  are  somewhat  nearer  the  violet  end  of 
the  spectrum.  Add  some  Stokes'  reagent  to  the  solution  and  again 
examine  spectroscopically.  Note  that  the  position  and  intensity  of  the 
absorption-bands  remain  unaltered. 


Fig.  64. — Diagram  of  Angular- vision  Spectroscope.  iLong.) 
The  white  light  F  enters  the  collimator  tube  through  a  narrow  slit  and  passes  to  the  prism, 
P,  which  has  the  power  of  refracting  and  dispersing  the  light.  The  rays  then  pass  to  the 
double  convex  lens  of  the  ocular  tube  and  are  deflected  to  the  eye-piece  E.  The  dotted 
lines  show  the  magnified  virtual  image  which  is  formed.  The  third  tube  contains  a  scale 
whose  image  is  reflected  into  the  ocular  and  shown  with  the  spectrum.  Between  the  light 
F  and  the  collimator  slit  is  placed  a  cell  to  hold  the  solution  undergoing  examination. 

The  following  is  a  delicate  chemical  test  for  the  detection  of  carbon 
monoxide  haemoglobin : 

Tannin  Test. — Divide  the  blood  to  be  tested  into  two  portions  and 
dilute  each  with  four  volumes  of  distilled  water.     Place  the  diluted  blood 


2i8  PHYSIOLOGICAL    CHEMISTRY. 

mixtures  in  two  small  flasks  or  large  test-tubes  and  add  20  drops  of  a  10 
per  cent  solution  of  potassium  ferricyanide/  Allow  both  solutions  to 
stand  for  a  few  minutes,  then  stopper  the  vessels  and  shake  one  vigorously 
for  10-15  minutes,  occasionally  removing  the  stopper  to  permit  air  to 
enter  the  vessel.^  Add  5-10  drops  of  ammonium  sulphide  (yellow)  and 
10  c.c.  of  a  10  per  cent  solution  of  tannin  to  each  flask.  The  contents  of 
the  shaken  flask  will  soon  exhibit  the  formation  of  a  dirty  olive  green 
precipitate,  whereas  the  flask  which  was  not  shaken  and  which,  therefore, 
still  contains  carbon  monoxide  haemoglobin,  will  exhibit  a  bright  red 
precipitate,  characteristic  of  carbon  monoxide  haemoglobin.  This 
test  is  more  delicate  than  the  spectroscopic  test  and  serves  to  detect 
the  presence  of  as  low  a  content  as  5  per  cent  of  carbon  monoxide 
haemoglobin. 

4.  Neutral  Methaemoglobin. — Dflute  a  little  defibrinated  blood 
(i  :  10)  and  add  a  few  drops  of  a  freshly  prepared  10  per  cent  solution  of 
potassium  ferricyanide.  Shake  this  mixture  and  observe  that  the  bright 
red  color  of  the  blood  is  displaced  by  a  brownish  red.  Now  dilute  a 
little  of  this  solution  and  examine  it  spectroscopically.  Note  the  single, 
very  dark  absorption-band  lying  to  the  left  of  D,  and,  if  the  dilution  is 
sufficiently  great,  also  observe  the  two  rather  faint  bands  lying  between 
D  and  E  in  somewhat  similar  positions  to  those  occupied  by  the  absorp- 
tion bands  of  oxy haemoglobin.  Add  a  few  drops  of  Stokes'  reagent  to  the 
methaemoglobin  solution  while  it  is  in  position  before  the  spectroscope 
and  note  the  immediate  appearance  of  the  oxyhaemoglobin  spectrum 
which  is  quickly  followed  by  that  of  haemoglobin. 

5.  Alkaline  Methaemoglobin. — Render  a  neutral  solution  of  met- 
haemoglobin, such  as  that  used  in  the  last  experiment  (4),  slightly  alkaline 
with  a  few  drops  of  ammonia.  The  solution  becomes  redder  in  color, 
due  to  the  formation  of  alkaline  methaemoglobin  and  shows  a  spectrum 
different  from  that  of  the  neutral  body.  In  this  case  we  have  a  band  on 
either  side  of  D,  the  one  nearer  the  red  end  of  the  spectrum  being  much 
the  fainter.  A  third  band,  darker  than  either  of  those  mentioned,  lies 
between  D  and  E  somewhat  nearer  E. 

6.  Alkali  Haematin. — Observe  the  spectrum  of  the  alkali  haematin 
prepared  in  Experiment  16  on  page  212.  Also  make  a  spectroscopic 
examination  of  a  freshly  prepared  alkali  haematin.^  The  typical  spec- 
trum of  alkali  haematin  shows  a  single  absorption-band  lying  across  D 
and  mainly  toward  the  red  end  of  the  spectrum. 

'  This  transforms  the  oxyha.-moglobin  into  mc-tha-moglobin. 

^  This  is  done  to  free  the  blood  from  carbon  monoxide  haemoglobin. 

"  Alkali  htematin  may  be  prepared  by  mixing  one  volume  of  a  concentrated  potassium 
hydroxide  or  sodium  hydroxide  solution  and  two  volumes  of  dilute  (1:5)  defibrinated  blood. 
This  mixture  should  be  heated  gradually  almost  to  boiling,  then  cooled  and  shaken  for  a 
few  moments  in  the  air  before  examination. 


HLOOD    AM)    l.VMFH.  219 

7.  Reduced  Alkali  Haematin  or  Haemochromogen. — Dilute  the 
alkali  haematin  solution  used  in  the  last  exj)eriment  (6)  to  such  an  extent 
that  it  shows  no  absorption  band.  Now  add  a  few  drops  of  Stokes' 
reagent  and  note  that  the  greenish-brown  color  of  the  alkali  haematin 
solution  is  displaced  by  a  bright  red  color.  This  is  due  to  the  formation 
of  hsemochromogen  or  reduced  alkali  hcTmatin.  Examine  this  solution 
spectroscopically  and  observe  the  narrow,  dark  absorption-ljand  lying 
midway  betweCVi  D  and  E.  If  the  dilution  is  not  too  great  a  faint  band 
may  be  observed  in  the  green  extending  across  E  and  I). 

8.  Acid  Haematin. — To  some  delibrinated  blood  add  half  its  vol- 
ume of  glacial  acetic  acid  and  an  equal  volume  of  ether.  Mix  thor- 
oughly. The  acidified  ethereal  solution  of  haematin  rises  to  the  top  and 
may  be  poured  off  and  used  for  the  spectroscopic  examination.  If 
desired  it  may  be  diluted  with  acidified  ether  in  the  ratio  of  one  part  of 
glacial  acetic  acid  to  two  parts  of  ether.  A  distinct  absorption-band  will 
be  noted  in  the  red  between  C  and  D  and  lying  somewhat  nearer  C  than 
the  band  in  the  methaemoglobin  spectrum.  Between  D  and  F  may  be 
seen  a  rather  indistinct  broad  band.  Dilute  the  solution  until  this  band 
resolves  itself  into  two  bands.  Of  these  the  more  prominent  is  a  broad, 
dark  absorption-band  lying  in  the  green  between  b  and  F.  The  second, 
a  narrow  band  of  faint  outline,  lies  in  the  light  green  to  the  red  side 
of  E.  A  fourth  very  faint  band  may  be  observed  lying  on  the  violet 
side  of  D. 

9.  Acid  Haematoporphyrin. — To  5  c.c.  of  concentrated  sulphuric 
acid  in  a  test-tube  add  two  drops  of  blood,  mixing  thoroughly  by  agitation 
after  the  addition  of  each  drop.  A  wine-red  solution  is  produced.  Ex- 
amine this  solution  spectroscopically.  Acid  haematoporphyrin  gives  a 
spectrum  with  an  absorption-band  on  either  side  of  D,  the  one  nearer  the 
red  end  of  the  spectrum  being  the  narrower. 

10.  Alkaline  Haematoporphyrin. — Introduce  the  acid  haemato- 
porphyrin solution  just  examined  into  an  excess  of  distilled  water.  Cool 
the  solution  and  add  potassium  hydroxide  slowly  until  the  reaction  is  but 
slightly  acid.  A  colored  precipitate  forms  which  includes  the  principal 
portion  of  the  haematoporphyrin.  The  presence  of  sodium  acetate 
facilitates  the  formation  of  this  precipitate.  Filter  off  the  precipitate  and 
dissolve  it  in  a  small  amount  of  dilute  potassium  hydroxide.  Alkaline 
haematoporphyrin  prepared  in  this  way  forms  a  bright  red  solution  and 
possesses  four  absorption-bands.  The  first  is  a  very  faint,  narrow  band 
in  the  red,  midway  between  C  and  D;  the  second  is  a  broader,  darker 
band  lying  across  D,  principally  to  the  violet  side.  The  third  absorption- 
band  lies  principally  between  D  and  E,  extending  for  a  short  distance 
across  E  to  the  violet  side,  and  the  fourth  band  is  broad  and  dark  and  lies 


220 


PHYSIOLOGICAL   CHEMISTRY. 


between  b  and  F.     The  first  band  mentioned  is  the  faintest  of  the  four 
and  is  the  first  to  disappear  when  the  solution  is  diluted. 

VII.  Instruments  Used  in  the  Clinical  Examination  of  the  Blood. 

I.  Fleischl's  Haemometer  (Fig.  65,  below). — This  is  an  instrument 
used  quite  extensively  clinically,  for  the  quantitative  determination  of 
hemoglobin.  The  instrument  consists  of  a  small  cylinder  which  is  pro- 
vided with  a  fixed  glass  bottom  and  a  movable  glass  cover,  and  which  is 
divided,  by  means  of  a  metal  septum,  into  two  compartments  of  equal 
capacity.     This  cylinder  is  supported  in  a  vertical  position  by  means  of  a 

mechanism  which  resembles  the  base  and 
stage  of  an  ordinary  microscope.  Under- 
neath the  stage  is  placed  a  colored  glass 
wedge  (see  Fig.  67,  p.  221),  so  arranged 
as  to  run  immediately  beneath  the  glass 
bottom  of  one  of  the  compartments  of  the 
cylinder  and  ground  in  such  a  manner 
that  each  part  of  the  wedge  corresponds 
in  color  to  a  solution  of  haemoglobin  of 
some  definite  percentage.  The  glass 
wedge  is  held  in  a  metal  frame  and  may 
be  moved  backward  or  forward  by  means 
of  a  rack  and  pinion  arrangement.  A 
scale  along  the  side  of  this  frame  indicates  the  percentage  of  the  normal 
amount  of  haemoglobin  which  each  particular  variation  in  the  depth  of 
color  of  the  ground  wedge  represents,  taking  the  normal  haemoglobin 
content  as  100.'^  In  a  position  corresponding  to  the  position  of  the 
mirror  on  the  ordinary  microscope  is  attached  a  light-colored  opaque 
plate  which  serves  to  reflect  the  light  upward  through  the  colored  wedge 
and  the  cylinder  to  the  eye  of  the  observer. 

In  making  a  determination  of  the  percentage  of  haemoglobin  by 
this  instrument  the  procedure  is  as  follows:  Fill  each  compartment 
about  three-fourths  full  of  distilled  water.  Puncture  the  finger-tip 
or  lobe  of  the  ear  of  the  subject  by  means  of  a  sterile  needle  or  scalpel  and, 
as  soon  as  a  drop  of  blood  appears,  place  one  end  of  the  capillary  pipette 
(Fig.  66),  which  accompanies  the  instrument,  against  the  drop  and  allow 
it  to  fill  by  capillary  attraction.  To  prevent  the  blood  from  adhering  to 
the  exterior  of  the  tube,  and  so  render  the  determination  inaccurate,  it  is 
customary  to  apply  a  very  thin  coating  of  mutton  fat  to  the  outer  surface 
before  using  or  to  wrap  the  tube  in  a  piece  of  oily  chamois  when  not  in 
use.     As  soon  as  the  tube  has  been  accurately  filled  with  blood  it  should 

*  The  scale  of  the  ordinary  instrument  is  usually  too  high. 


Fig.  65. 


-Fleischl's  H^mometer. 
{Da  Costa.) 


BLOOD  AND    LYMPH. 


221 


Fig.  66. — Pipette 
OF      Fleischl's 

H^MOMETER. 


be  dipped  into  the  water  of  one  of  the  compartments  of  the  cylinder  and 
all  traces  of  the  blood  washed  out  with  water  by  means  of  a  small  dropper 
which  accompanies  the  instrument.  If  the  blood  is  not  well  distributed 
throughout  the  compartment  and  docs  not  form  a  homogeneous  solution 
the  contents  of  the  compartment  should  be  mixed  thoroughly  by  means 
of  the  metal  handle  of  the  capillary  measuring  pipette.  When  this  has 
been  done  eacli,  compartment  should  be  completely  filled  with  distilled 
water  and  the  glass  cover  adjusted,  care  being  taken 
that  the  contents  of  the  two  compartments  do  not  mix. 
Now  adjust  the  cylinder  so  that  the  compartment 
containing  the  pure  distilled  water  is  immediately 
above  the  colored  glass  wedge.  By  means  of  the  rack 
and  pinion  arrangement  manipulate  the  colored  wedge 
until  a  portion  of  it  is  found  which  corresponds  in 
color  with  the  diluted  blood.  When  this  agreement 
in  color  has  been  secured  the  point  on  the  scale  cor- 
responding to  this  particular  color  should  be  read  and  the  actual  per- 
centage of  hcxmoglobin  computed.  For  instance,  if  the  scale  reading  is 
90  it  means  that  the  blood  under  examination  contains  90  per  cent  of  the 
normal  quantity  of  haemoglobin,  i.  e.,  90  per  cent  of  14  per  cent. 

2.  Fleischl-Miescher  Haemometer. — The  apparatus  of  Fleischl 
has  recently  been  modified  by  Miescher.  If  all  precautions  are  taken, 
the  margin  of  error  in  the  absolute  quantity  of  haemoglobin  determined 
by  this  instrument  does  not  exceed  0.15-0.22  per  cent  by  weight  of  the 
blood.  Detailed  directions  for  the  manipulation  of  the  Fleischl-Miescher 
haemometer  accompany  the  instrument.  In  brief  Miescher  modified  the 
instrument  as  follows:  (i)  The  scale  of  each  instrument  is  supplied  with 

a  caliber  table  of  absolute  haemo- 
globin values,  expressed  in  milli- 
grams: the  scale  of  Fleischl's  haemo- 
meter shows  the  percentage  of 
haemoglobin  in  relation  to  an 
average  selected  somewhat  arbi- 
trarily. Thus  many  errors  arising 
from  the  irregular  coloring  of  the 
glass  wedge  of  the  older  apparatus  are  avoided  in  the  instrument  as 
modified.  (2)  Each  instrument  is  accompanied  by  a  measuring  pipette 
(melangeur)  which  allows  of  a  more  accurate  measurement  of  the  blood 
than  was  possible  with  the  capillary  tubes  of  the  older  apparatus.  (3) 
With  the  aid  of  the  measuring  pipette  mentioned  above  blood  of  varying 
degrees  of  concentration  may  be  compared.  In  this  way  the  individual 
examinations  are  controlled  and  a  check  upon  the  accuracy  of  the  gradu- 


FiG.    67. — Colored    Glass    Wedge    of 
Fleischl's  H^mometer.     {Da  Cosla.) 


222 


PHYSIOLOGICAL    CHEMISTRY. 


ation  in  the  color  of  the  glass  wedge  is  also  afforded.  This  wedge  is  much 
more  evenly  and  accurately  colored  than  in  the  unmodified  apparatus 
of  Fleischl.  (4)  Before  reading  the  percentage  as  indicated  by  the  scale, 
the  chamber  is  covered  with  a  glass  and  a  diaphragm  which  sharply 
define  the  field  on  all  sides  without  the  formation  of  a  meniscus. 

The  measuring  pipette  is  constructed  essentially  the  same  as  the 
pipettes  which  accompany  the  Thoma-Zeiss  apparatus  (see  page  225). 

The  capillary  portion,  however,  is 
graduated,  i,  2/3  and  1/2  which 
enables  the  observer  to  dilute  the 
blood  sample  in  the  proportion  of 
1:200,  1:300  or  1:400  as  he  may 
desire.  If  there  is  difficulty  in 
drawing  in  the  blood  exactly  to  one 
of  the  graduations  just  mentioned 
the  amount  of  blood  above  or  below 
the  volume  indicated  by  the  gradu- 
ation may  be  determined  by  means 
of  certain  delicate  cross-lines  which 
are  placed  directly  above  and  below 
the  graduation.  Each  cross-line 
corresponds  to  i/ioo  of  the  volume 
of  the  capillary  tube  from  the  tip 
to  the  I   graduation. 

A  o.  I  per  cent  solution  of  sodium 

carbonate  is  used  to  dissolve  the 

stroma  of  the  erythrocytes  and  so 

render  the  blood  solution  perfectly 

clear.     If  this  is  not  done  the  color 

of   the   blood    solution    invariably 

appears  darker  in  tone  than  that  of 

the  colored  glass  wedge.     A  freshly 

prepared  sodium  carbonate  solution  should  be  used  in  order  that  the 

clearness  of  the  solution  may  not  be  marred  by  the  presence  of  sodium 

bicarbonate. 

3.  Dare's  Haemoglobinometer  (Fig.  68). — This  instrument,  as 
the  name  signifies,  is  used  for  the  determination  of  haemoglobin.  In 
using  either  Fleischj's  haemometer  or  the  instrument  as  modified  by 
Miescher  the  blood  is  diluted  for  examination,  whereas  with  the  Dare 
instrument  no  dilution  is  required.  This  probably  allows  of  rather 
more  accurate  determinations  than  are  possible  with  the  old  Fleischl 
apparatus. 


-Dare's  H^moglobinometer. 
{Da  Costa.) 
R,  Milled  wheel  acting  by  a  friction  bear- 
ing on  the  rim  of  the  color  disc;  S,  case  in- 
closing color  disc,  and  provided  with  a  stage 
to  which  the  blood  chamber  is  fitted ;  T,  mov- 
able wing  which  is  swung  outward  during  the 
observation,  to  serve  as  a  screen  for  the  ob- 
server's eyes,  and  which  acts  as  a  cover  to 
inclose  the  color  disc  when  the  instrument  is 
not  in  use;  U,  telescoping  camera  tube,  in 
position  for  examination;  V,  aperture  admit- 
ting light  for  illumination  of  the  color  disc;  X, 
capillary  blood  chamber  adjusted  to  stage  of 
instrument,  the  slip  of  opaque  glass,  W,  being 
nearest  to  the  source  of  light;  Y,  detachable 
candle-holder;  Z,  rectangular  slot  through 
which  the  haemoglobin  scale  indicated  on  the 
rim  of  the  color  disc  is  read. 


BLOOD   AND    LYMPH. 


223 


The  instrument  consists  essentially  of  the  following  parts:  (i)  A 
capillary  observation  cell,  (2)  a  semicircular  colored  glass  wedge,  (3) 
a  milled  wheel  for  manipulating  the  wedge,  (4)  a  candle  used  to  illuminate 
portions  of  the  capillary  observation  cell  and  the  colored  wedge,  (5)  a 
small  telescope  used  in  the  examination  of  the 
areas  illuminated  by  the  candle  flame,  (6)  a 
scale  graduated  in  percentages  of  the  normal 
amount  of  haemoglobin,  (7)  a  hard-rubber 
case,  (8)  a  movable  screen  attached  to  the  case. 

The  capillary  observation  cell  is  formed  of 
two  small,  polished  rectangular  plates  of  glass, 
one  being  transparent  and  the  other  opaque. 
When  held  in  position  on  the  instrument,  by 
means  of  a  small  metal  bracket,  the  opaque 
portion  of  the  cell  is  nearer  the  candle  and 
thus  serves  to  soften  the  glare  of  light  when 


an  observation  is  being  made.      The  trans- 


FiG.  69. — Horizontal  Sec- 
tion OF  Dare's  H^emoglo- 
BiNOMETER.     {Da  Cosia.) 


parent  portion  of  the  cell  is  directly  over  a 
circular  opening  in  the  case,  through  which 
the  blood  specimen  is  viewed  by  means  of  the  small  telescope. 

The  semicircular  colored  glass  wedge  is  so  ground  that  each  par- 
ticular shade  of  color  corresponds  to  that  possessed  by  fresh  blood  which 
contains  some  definite  percentage  of  haemoglobin.  It  is  mounted  upon 
a  disc  which  may  be  manipulated  by  the  milled  wheel  in  such  a  manner 
as  to  bring  successive  portions  of  the  wedge  in  position  to  be  viewed 
through  a  circular  opening  contiguous  to  the  opening  through  which  the 


Fig.  70.— Method  of  Filling  the  Capillary  Observation  Cell  of  Dare's  Hj:mo- 

GLOBINOMETER.       (Da  Cosla.) 

blood  specimen  is  viewed.     For  a  further  description  of  the  instrument 
see  Figs.  68,  69,  and  70. 

In  using  the  Dare  haemoglobinometer  proceed  as  follows:  Puncture 
the  tmger-tip  or  lobe  of  the  ear  of  the  subject  by  means  of  a  needle  or 
scalpel  and,  after  a  drop  of  blood  of  good  proportions  has  formed,  place 


224 


PHYSIOLOGICAL   CHEMISTRY. 


the  flat  capillary  observation  cell  in  contact  with  the  drop  and  allow  it  to 
fill  by  capillary  attraction  (Fig.  70) .  Replace  the  cell  in  its  proper  place  on 
the  instrument.  When  in  position,  a  portion  of  this  cell  may  be  observed 
through  a  small  telescope  attached  to  the  apparatus.  It  is  viewed 
through  a  circular  opening  and  near  this  circle  is  a  second  one  through 
which  a  portion  of  a  semicircular  colored  glass  wedge  is  visible.  These 
two  circles  are  illuminated  simultaneously  by  means  of  the  flame  of  a 
candle.  The  colored  glass  may  be  rotated  by  means  of  a  milled  wheel 
and  the  point  of  agreement  of  the  color  of  the  adjoining  discs  may  be 
determined  in  the  same  way  as  in  Fleischl's  haemometer.  The  scale 
reading  gives  the  percentage  of  the  normal  quantity  of  haemoglobin  which 
the  blood  sample  under  examination  contains.  Compute  the  actual 
haemoglobin  contfent  in  the  same  manner  as  from  the  scale  reading  of  the 
Fleischl  haemometer  (see  page  221). 

4.  Tallquist's  Haemoglobin  Scale. — This  consists  essentially  of 
a  series  of  ten  colors  corresponding  to  stains  produced  by  blood  con- 
taining varying  percentages  of  haemoglobin.  In  using  this  scale  a  drop 
of  blood  is  allowed  to  fall  on  a  small  section  of  filter  paper  and  the  resulting 
color  is  compared  with  the  ten  colors  of  the  scale.  When  the  color  in  the 
scale  is  found  which  corresponds  to  the  color  of  the  blood  stain  the  ac- 
companying haemoglobin  value  is  read  off  directly.  This  is  a  very  con- 
venient method  for  determining  haemoglobin  at  the  bedside.  There 
is  a  possibility  of  the  colors  being  inaccurately  printed,  however,  and 
even  if  originally  correct  in  tint,  under  the  continued  influence  of  air  and 
light  they  must  eventually  alter  somewhat. 


Fig.  71. — Thoma-Zeiss  Counting  Chamber.     (Da  Costa.) 


5.  Thoma-Zeiss  Haemocytometer. — This  is  an  instrument  used 
in  "blood  counting,"  i.  e.,  in  determining  the  number  of  erythrocytes 
and  leucocytes.  The  instrument  consists  of  a  microscopic  slide  con- 
structed of  heavy  glass  and  provided  with  a  central  counting  cell  (see 
Fig.  71,  below).  This  cell,  with  the  cover  glass  in  position,  is  exactly 
0.1  millimeter  deep.     The  floor  of  the  cell  is  divided  by  delicate  lines  into 


BLOOD  AND    LYMPH. 


22i 


U 


squares  each  of  which  is  i  400  of  a  square  millimeter  in  area  (see  Fig. 
73,  page  22O).  The  volume  of  blood  therefore  between  any  particular 
square  and  the  cover  glass  above  must  be  1/4000  cubic  millimeter.  Ac- 
companying each  instrument  are  two  capillary  i)ipetles  (Fig.  72.  below), 
each  constructed  with  a  mixing  bulb  in  its  upper  por- 
tion. Each  bulb  is  further  provided  with  an  enclosed 
glass  bead  which  is  of  great  assistance  in  mixing  the 
contents  of  the  chamber.  The  stem  of  each  pipette  is 
graduated  in  tenths  from  the  tip  to  the  bulb.  The 
final  graduation  at  the  upper  end  of  the  bulb  is  10 1 
on  the  pipette  used  in  mixing  the  blood  sample  in 
which  the  erythrocytes  are  counted  (erythrocytom- 
eter,  see  Fig.  72,  page  225),  and  11  on  the  pipette 
used  in  mixing  the  blood  sample  for  the  leucocyte 
count  (leucocytometer,  see  Fig.  72,  page  225).  In 
making  ''blood  counts"  with  the  haemocytometer  it  is 
necessary  to  use  some  diluting  fluid.  Two  very  satis- 
factory forms  of  fluid  for  this  purpose  are  Toison's 
and  Sherrington's  solutions.^  When  either  of  these 
solutions  is  used  as  the  diluting  fluid  it  is  possible  to 
make  a  very  satisfactory  count  of  both  the  erythro- 
cytes and  leucocytes  from  the  same  preparation,  since 
the  leucocytes  are  stained  by  the  methyl-violet  or 
methylene-blue. 

In  counting  the  erythrocytes  by  means  of  the  haemo- 
cytometer, proceed  as  follows:  Thoroughly  cleanse  the 
tip  of  the  finger  or  lobe  of  the  car  of  the  subject  by 
the  use  of  soap  and  water,  alcohol  and  ether  applied 
in  the  sequence  just  given.  Puncture  the  skin  by 
means  of  a  needle  or  scalpel  and  allow  the  blood  drop 
to  form  without  pressure.  Place  the  tip  of  the  pipette 
in  contact  with  the  blood  drop,  being  careful  to  avoid  touching  the 
skin,  and  draw  blood  into  the  pipette  up  to  the  point  marked  0.5  or  1 
according  to  the  desired  dilution.  Rapidly  wipe  the  tip  of  the  pipette  and 
immediately  fill  it  to  the  point  marked  10 1  with  Toison's  or  Sherrington's 
solution.  Now  thoroughly  mix  the  blood  and  diluting  fluid  within  the 
mixing  chamber  by  tapping  the  pipette  gently  against  the  finger,  or  by 


V 


B 

Fig.  72. — Thoma- 
Zeiss  Capillary 
Pipettes. 

A,Erythrocytometer; 
B,    Leucocytometer. 


'  Toison"<    solution    has  the    following 
formula: 

Methyl-violet 0.025  gram. 

Sodium  chloride i  gram. 

Sodium  sulphate 8  grams. 

Glycerol 30  grams. 

Distilled  water i6o  grams. 


Sherrington's  solution  has  the  follo\Aing 
formula : 

Methylene-blue o .  i  gram. 

Sodium  chloride 1.2  gram. 

Neutral  potassium  oxalate....     i  .2  gram. 
Distilled  water 300.0  grams. 


226 


PHYSIOLOGICAL    CHEMISTRY. 


shaking  it  while  held  securely  with  the  thumb  at  one  end  and  the  middle 
finger  at  the  other.  After  the  two  fluids  have  been  thoroughly  mixed  the 
diluting  fluid  contained  in  the  capillary-tube  below  the  bulb  should  be 
discarded  in  order  to  insure  the  collection  of  a  drop  of  the  thoroughly 
mixed  blood  and  diluting  solution  for  examination.  Transfer  a  drop  from 
the  pipette  to  the  ruled  floor  of  the  counting  chamber  and,  after  placing 
the  cover  glass  firmly  in  position/  allow  an  interval  of  a  few  minutes  to 
elapse  for  the  corpuscles  to  settle  before  making  the  count.  Now  place 
the  slide  under  the  microscope  and  count  the  number  of  erythrocytes 
in  a  number  of  squares,  counting  the  corpuscles  which  are  in  contact 


Fig.    7.- 


-Ordinary   Ruling   of  Thoma-Zeiss   Counting   Chamber.     {Da  Costa.) 


with  the  upper  and  the  right-hand  boundaries  of  the  square  as  belonging 
to  that  square.  Take  the  squares  in  some  definite  sequence  in  order  that 
the  recounting  of  the  same  corpuscles  may  be  avoided.  A  satisfactory 
procedure  is  to  begin  in  the  upper  right-hand  corner  and  proceed  from 
left  to  right  counting  the  cells  in  each  individual  square.  Take  the  next 
lower  row  of  squares  and  count  from  left  to  right  and  so  on  (see  Fig.  77, 
p.  232).  Of  course,  all  things  being  equal,  the  greater  the  number  of 
squares  examined  the  more  accurate  the  count.  It  is  considered  essen- 
tial under  all  circumstances,  where  an  accurate  count  is  desired  that 
the  counting  chamber  shall  be  filled,  at  least  twice,  and  the  individual 
counts  made  in  each  instance,  as  indicated  above,  before  the  data  are 
deemed  satisfactory.  Under  no  conditions  should  less  than  200  squares 
be  examined. 

To  calculate  the  number  of  erythrocytes   per   cubic   millimeter   of 
undiluted  blood  proceed  as  follows:  Determine  the  number  of  corpuscles 

'  If  the  cover  glass  is  in  accurate  apposition  to  the  counting  cell  Newton's  rings  may  be 
plainly  observed. 


BLOOD   AND    LYMPH. 


227 


in  any  given  number  of  squares  and  divide  this  total  by  the  number  of 
squares,  thus  obtaining  the  average  number  of  erythrocytes  per  square. 
Multiply  this  average  by  4,000  to  obtain  the  number  of  erythrocytes 
per  cubic  millimeter  of  diluted  blood,  and  multiply  this  product  by  100 
or  200,  according  to  the  dilution,  to  obtain  the  number  of  erythrocytes 
per  cubic  millimeter  of  undiluted  blood.     Thus: 


Average   number  .,of   erythrocytes 
per  square 


^,  ,  N       Xumher   of   erythrocytes    per 

X  4,000  X  200  (or  100)=         ,.        .,,.      .-  ■'         ' 

^'  ^  cubic  milhmeler. 


Great  care  should  be  taken  to  see  that  the  capillary  pipette  is  prop- 
erly cleaned.     After  using,  it  should  be  immediately  rinsed  out  with  the 


Fig.  74. — Zappert's  Modified  Ruxing  of  Thoma-Zeiss  Cox.'xting  Chamber.     {Da  Costa.) 


diluting  fluid,  then  with  water,  alcohol,  and  ether  in  the  sequence  given. 
Finally  dry  air  should  be  drawn  through  the  capillary  and  a  horse  hair 
inserted  to  prevent  the  entrance  of  dust  particles. 

In  counting  leucocytes  by  means  of  the  haemocytometer  proceed  as 
follows:  As  mentioned  above,  if  the  diluting  fluid  is  either  Toison's  or 
Sherrington's  solution  the  leucocytes  may  be  counted  in  the  same  specimen 
of  blood  in  which  the  erythrocytes  are  counted.  When  this  is  done  it 
is  customary  to  use  a  slide  provided  with  Zappert's  modified  ruling 
(Fig.  74,  above).  This  method  is  rather  more  accurate  than  the  older 
one  of  counting  the  leucocytes  in  a  separate  specimen  of  blood.  Further- 
more, it  is  obviously  preferable  to  count  both  the  erythrocytes  and  the 
leucocytes  from  the  same  blood  sample.  To  insure  accuracy  the  number 
of  leucocytes  within  the  whole  ruled  region  should  be  determined  in 
duplicate  blood  samples.  This  includes  the  examination  of  an  area  eigh- 
teen times  as  great  as  the  old  style  Thoma-Zeiss  central  ruling.     This 


228  PHYSIOLOGICAL    CHEMISTRY. 

region  then  would  correspond  to  3,600  of  the  small  squares  and,  if  duplicate 
examinations  were  made,  the  total  number  of  small  squares  examined 
would  aggregate  7,200.     The  calculation  would  be  as  follows : 

Number    of    leucocytes    in    7,200   ^.        ^.  Number  of  leucocytes  per  cubic 

'  X200X4, 000-^7,200=  .,,.      ,  '         ^ 

squares  ^  '  millimeter. 

If  a  Zappert  slide  is  not  available,  a  good  plan  to  follow  is  to  place  a 
diaphragm  in  the  tube  of  the  ocular  of  the  microscope  consisting  of  a  circle 
of  black  cardboard  or  metaP  having  a  square  hole  in  the  center  of  such 
a  size  as  to  allow  of  the  examination  of  exactly  100  squares  or  one-fourth  of 
a  square  millimeter  at  one  time.  With  this  arrangement  any  portion  of 
the  specimen  may  be  examined  and  counted  whether  wdthin  or  without  the 
ruled  area.  In  counting  by  means  of  this  device  it  is,  of  course,  helpful 
if  the  microscope  is  provided  with  a  mechanical  stage,  but  even  without 
this  arrangement,  if  the  observer  is  careful  to  see  that  the  leucocytes  at 
the  extreme  boundary  of  one  field  move  to  the  opposite  boundary  when  the 
position  of  the  slide  is  changed,  the  device  may  be  very  satisfactorily  em- 
ployed. The  leucocytes  should  be  counted  in  36  of  the  diaphragm- 
fields  in  duplicate  specimens  and  the  calculation  made  in  the  same  manner 
as  explained  above. 

If  the  leucocytes  are  counted  in  a  separate  specimen  of  blood  ordinarily 
the  diluting  fluid  is  0.3-0.5  per  cent  acetic  acid,  a  fluid  in  which  the  leuco- 
cytes alone  remain  visible.  Under  these  conditions  the  dilution  is 
customarily  made  in  the  pipette  having  11  as  the  final  graduation.  The 
capillary  portion  is  of  larger  caliber  and  so  rec^uires  a  greater  amount  of 
blood  to  fill  it  to  the  0.5  or  i  mark  than  is  recjuired  in  the  use  of  the  other 
form  of  pipette.  In  counting  the  leucocytes  according  to  this  method  it 
is  customary  to  draw  blood  into  the  pipette  up  to  the  i  mark  and  immedi- 
ately fill  the  remaining  portion  of  the  apparatus  to  the  1 1  graduation  with 
the  0.3-0.5  per  cent  acetic  acid.  It  then  remains  to  count  the  number  of 
leucocytes  in  the  whole  central  ruled  portion  of  400  squares.  This 
should  be  done  in  duplicate  samples  and  the  calculation  made  as  follows: 

Number    of    leucocytes    in    Soo   .  .  ^,  „  Number    of    leucocytes    per    cubic 

■'  X  4,000  X  10-^-800=  .,,.       .  -  ' 

squares  millimeter. 

6.  Biirker's  Haemocytometer.- — This  is  an  improved  apparatus^  for 
the  more  accurate  counting  of  erythrocytes  than  is  possible  by  the 
Thoma-Zeiss  apparatus.  The  principles  involved  arc  somewhat  dif- 
ferent from  those  in  force  with  the  latter  apparatus.  For  example,  the 
blood  is  diluted  in  a  separate  vessel,  not  in  the  pipette  with  which  the 
sample  is  drawn,  and  furthermore  the  cover  glass  is  applied  to  the  counting 

'  Ehrlich's  mechanical  eye-piece  with  iris  diaphragm  is  also  yery  satisfactory  for  this 
purpose. 

^  Biirker:  Pfliiger's  Archiv.,  142,  337,  191 1;  Miinch.  med.  Woch.,  59,  pp.  14  and  89,  1912. 
'  Manufactured  by  C.  Zeiss,  Jena. 


BLOOD    AM)    I.V.MPH. 


229 


chamber  and  clamped  in  place  before  the  diluted  blood  is  applied  to  the 
ruled  area.  Hayem's  solution'  is  used  as  the  diluting  fluid.  Toison'r- 
solution  is  not  satisfactory  for  use  with  the  Hiirker  counting  chamber  as 
its  viscosity  is  too  great.  The  corpuscles  settle  rapidly  in  Hayem's  fluid 
as  the  specific  gravity  of  the  fluid  is  1015  whereas  that  of  the  erythrocytes 
is  iO()o. 

The  pipet^te   for  measuring  the  cjuantily  of  blood    (F'ig.   75,   uj)per 
pipette)   has  a  point  which  is  not  ground  dull  but  is  polished.     This 


Fig.  75. — Burker's  1'ipkttks,  Mixing  Flasks  and  C  )inting  Chamber. 

allows  of  better  judgment  in  deciding  whether  the  column  of  blood  ex- 
tends to  the  very  tip.  The  volume  of  the  pipette  between  tip  and  mark 
is  25  cubic  millimeters.  The  mark  extends  all  the  way  around  the  tube 
so  that  errors  of  parallax  may  be  avoided. 

The  pipette  for  measuring  the  diluting  fluid  (Fig.  75,  middle  pipette) 
also  has  a  polished  point  and  circular  mark  and  delivers  4975  cubic 
millimeters.  This  volume  of  diluting  fluid  with  25  cubic  millimeters  of 
blood  gives  a  dilution  of  i  :20c.  Both  pipettes  are  provided  with  a  piece 
of  rubber  tubing  and  mouth-piece. 

'  Hayem's  solution  has  the  following  formula: 

Mercuric  chloride 0.25  gram. 

Sodium  chloride 0.5    gram. 

Sodium  sulphate 2.5    grams. 

Distilled  water 100  c    yrams. 


230  PHYSIOLOGICAL   CHEMISTRY. 

For  transferring  the  diluted  blood  from  the  diluting  flask  to  the 
chamber  a  plain  pipette  provided  with  a  rubber  cap  is  used  (Fig.  75, 
lower  pipette).  It  is  filled  by  pressing  the  cap  slowly  with  the  index 
finger,  inserting  the  tip  into  the  lic|uid  and  then  releasing  the  pressure. 

The  diluting  is  done  in  a  small  round-bottomed  flask  as  shown  in 
Fig.  75.  Several  of  these  flasks  should  be  kept  on  hand  in  a  wooden 
rack  which  will  hold  them  in  an  upright  position.  Each  flask  is  provided 
with  a  parafi&ned,  or  smooth  cork  stopper. 

In  the  older  counting  chambers  the  floor  of  the  chamber  is  circular  and 
the  counting  is  done  in  the  center  of  this  space.  The  corpuscles  are 
therefore  counted  in  the  center  of  a  capillary,  circular  film  where  on 
account  of  surface  tension  their  number  is  slightly  greater  than  elsewhere. 
This  source  of  error  is  avoided  in  the  new  counting  chamber  (Fig.  78)  in 
which  the  floor  is  represented  by  the  upper  surface  of  a  piece  of  glass  25 
mm.  long  and  5  mm.  wide  which  is  rounded  off  at  both  ends  and  divided 
into  two  portions  by  a  groove  1.5  mm.  wide  through  the  center.  At  each 
side  of  this  floor  piece,  separated  from  it  by  a  groove  is  a  glass  plate 
(7.5  mm.  X21  mm.)  of  such  height  that  the  space  between  the  floor  of 
the  cell  and  a  cover  glass  placed  across  the  plates  is  o.ioo  mm.  A  cover 
glass  23  mm.  long  and  21  mm.  wide  with  rounded  polished  edges  is 
used  so  that  the  rounded  ends  of  the  floor  piece  project  beyond  it.  The 
chamber  is  provided  with  clamps  to  press  the  cover  glass  firmly  upon 
both  plates  (Fig.  75). 

The  ruling  on  each  portion  of  the  floor  piece  is  that  shown  in  Fig.  76, 
which  will  be  explained  below. 

Measuring  the  Diluting  Fluid. — Four  thousand  nine  hundred  and 
seventy-five  cubic  millimeters  of  diluting  fluid  (Hayem's)  are  measured 
out  into  the  diluting  flask.  To  do  this  the  pipette  is  filled  by  suction  to 
slightly  above  the  mark  and  the  rubber  tube  is  carefully  clamped  off. 
Then  with  a  soit  piece  of  linen  the  tip  is  wiped  dry.  The  meniscus  is 
then  accurately  adjusted  to  the  mark  by  lightly  touching  the  point  of  the 
pipette  to  the  cleaned  tip  of  the  finger.  The  pipette  is  then  inserted  into 
the  diluting  flask  and  with  the  tip  nearly  touching  the  bottom  of  the  flask 
the  fluid  is  allowed  to  run  out.  The  time  of  the  flow  should  be  about 
forty  seconds  and  is  controlled  by  placing  the  tip  of  the  index  finger 
loosely  upon  the  mouth  piece.  The  pipette  is  emptied  completely  by 
alternately  blowing  through  it  and  touching  it  to  the  wall  of  the  flask 
slightly  above  the  level  of  the  liquid.  The  drops  clinging  to  the  wall  are 
united  with  the  bulk  of  the  Hquid  by  a  suitable  motion  of  the  flask.  The 
flask  is  then  stoppered,  care  being  taken  from  now  on  that  none  of  the 
liquid  ever  touches  the  neck  of  the  flask  or  the  stopper. 

Taking  the  Blood  Sample. — Usually  the  best  time  to  draw  the  blood  is 


BLOOD   AND    LYMPH.  23 1 

before  breakfast.  For  a  single  determination  the  author  prefers  to  draw- 
it  from  the  tip  of  the  fourth  finger  of  the  left  hand.  For  repeated  deter- 
minations it  is  well  to  change  off  between  third,  fourth  and  fifth  fingers  of 
left  hand.  The  temperature  of  the  room  should  not  be  below  17°  C. 
to  prevent  an  undue  contraction  of  the  cutaneous  vessels.  The  instru- 
ment used  to  puncture  the  finger  should  have  a  chisel-shaped  point  which 
is  preferable  to  the  ordinary  lancet-shaped  point.  The  first  drop  of  blood 
is  wiped  off.  Into  the  second  one  the  tip  of  the  pipette  is  inserted  and 
blood  is  drawn  in  until  the  meniscus  is  even  with  or  a  little  beyond  the 
mark.  The  tip  is  then  wiped  off  without  touching  the  capillary  opening 
and  the  observer  assures  himself  that  the  column  of  blood  extends  to  the 
very  end  of  the  capillary.  The  meniscus  is  then  accurately  adjusted  to 
the  mark. 

Mixing  of  the  Blood  and  Diluting  Fluids. — The  tip  of  the  pipette  is  now 
dipped  into  the  diluting  fluid  which  has  been  measured  into  the  llask  and 
the  blood  is  slowly  blown  out.  The  blood  having  a  much  higher  specific 
gra%-ity  than  the  Hayem's  fluid  sinks  to  the  bottom.  The  pipette  is  then 
filled  with  the  pure  supernatant  diluting  fluid  and  emptied  again,  care 
being  taken  to  avoid  air  bubbles.  This  is  repeated  until  the  blood  is 
removed  as  completely  as  possible.  To  mix  the  blood  and  diluting  fluid 
the  flask  is  rotated  for  two  minutes  in  spiral  curves  of  continually  decreas- 
ing radius.  The  motion  should  be  alternately  clockwise  and  counter- 
clockwise. After  complete  mixing  the  pipette  is  rinsed  out  several  times 
with  the  diluted  blood. 

Transferral  of  the  Diluted  Blood  to  the  Chamber. — The  counting  cham- 
ber which  has  been  cleaned  with  distilled  water  and  alcohol-ether  and 
then  wiped  dry  with  a  soft  cloth  as  free  from  lint  as  possible  is  placed  upon 
a  black  surface  and  carefully  brushed  with  a  camel's  hair  brush.  The 
cover  glass  is  now  placed  over  the  chamber  by  sliding  it  over  the  two 
glass  plates  with  both  thumbs  while  the  index  fingers  are  pressing  it 
down.  By  means  of  the  clamps  it  is  held  in  place  firmly  so  that  Newton's 
rings  (if  possible  of  the  first  order:  brown  and  black)  may  be  seen  over 
the  entire  area  of  the  plates.  The  chamber  is  placed  upon  the  stage  of  the 
microscope  and  is  brought  into  a  horizontal  position. 

Before  transferring  the  diluted  blood  to  the  chamber  the  flask  must  be 
shaken  for  two  minutes  as  described  before.  The  liquid  shows  a  cloudy 
appearance  and  must  be  allowed  to  stand  until  the  turbidity  has  become 
uniform. 

One  of  the  plain  pipettes  described  above  is  now  inserted  into  the 
diluted  blood  while  slight  pressure  is  being  exerted  on  the  rubber  cap. 
The  pressure  is  released  slowly  and  the  liquid  rises  into  the  pipette.  The 
point  of  the  pipette  is  now  immediately  placed  upon  one  of  the  projecting 


232 


PHYSIOLOGICAL    CHEMISTRY. 


ends  of  the  floor  plate  and  very  slight  pressure  is  exerted  on  the  rubber 
cap  until  the  liquid  coming  from  the  pipette  just  reaches  the  cover  glass 
when  the  pressure  is  released.     An  instantaneous  filling  of  the  capillary 


Fig.  76. — Ruling  of  Bdrker  Counting  Chamber. 


\5  C 


5 

1 

3 

13 

^1 

1 

-^ 

— 

^ 

^5 

5 

-^ 

^ 

-» 

«-9 

9 

" 

-^ 

^ 

^ 

<^13 

13 

1 

5 

t 

J 

> 

I. 

J 

Fig.  77. — Schema. 

space  results.  The  pipette  should  be  emptied  immediately,  rinsed  with 
distilled  water  and  placed  in  an  upright  position  in  a  beaker  of  water. 
The  other  portion  of  the  counting  chamber  is  now  filled  in  the  same  way 


HI.OOI)    AM)    LYMPH. 


^2>3 


with  a  second  pipette  and  about  one  minute  is  allowed  for  the  settling  of 
the  corpuscles.  During  this  time  the  jjipettes  may  be  washed  with  dis- 
tilled water  and  ether-alcohol  and  dried  In-  suction.  Occasionally,  the 
pipettes  should  l)e  cleaned  with  a  horse  hair  and  with  concentrated  II.^SO^ 
containing  a  little  KXr.O^. 

To  see  whether  the  distribution  of  the  corpuscles  has  been  uniform 
the  chamber  is  Hluminated  with  a  wide-oj)en  diaj)hragm  and  viewed  at  an 
angle.  If  the  opacity  is  not  uniform  in  either  of  the  portions  of  the 
chamber,  that  one  should  not  be  used  for  counting.  If  the  counting  must 
be  interrupted  or  requires  a  longtime  a  moist  chamber'  should  be  used  to 
prevent  evaporation  of  the  diluting  fluid.  The  diluted  blood  may  be 
retained  in  the  mixing  flasks  and  duplicate  countings  obtained  after  the 
lapse  of  twenty-four  hours  or  niore  according  to  Biirker. 


Tiefe 

O.lOOmni. 

C.Zeiss 
Jerta 

kJ 

© 

O.Olmm. 

I        I 


Fig.  78. — BuRKER  Counting  Ch.\mber. 

Counting  and  Calculation. — A  mechanical  stage  movable  in  two  direc- 
tions is  indispensable.  With  a  magnification  of  320  diameters  the 
counting  is  begun  in  the  left  upper  corner  of  the  ruling.  Proceed  from 
left  to  right  along  one  row  then  move  from  right  to  left  along  the  next 
lower  row,  and  so  on.  Only  the  small  sc|uares  are  used  for  counting  (see 
Fig.  76),  and  the  figures  are  recorded  in  the  schema'-  (see  Fig.  77)  in  which 
the  squares  crossed  by  horizontal  or  vertical  lines  correspond  to  the  small 
squares  used  for  counting.  Usually  80  squares  are  counted  and  by 
recording  the  figures  in  the  schema  the  count  may  be  verified  and  an  idea 


'  Biirker:   Pfliiger's  Archiv,  iiS,  465,  IQ07. 

-  The  firm  of  II.  Laupp  in  Tubingen  has  put  this  schema  on  the  market  (in  pac  ks  of  100) 


234  PHYSIOLOGICAL   CHEMISTRY. 

of  the  uniformity  of  the  distribution  may  be  formed.  Half  of  the  counted 
squares  should  be  in  the  one,  half  in  the  other  portion  of  the  counting 
chamber.  For  more  accurate  measurements  more  squares  may  be 
counted. 

The  observer  will  do  well  not  to  attempt  counting  each  individual 
corpuscle  in  a  square.  After  some  practice  each  typical  group  of  cor- 
puscles will  immediately  suggest  a  number.  A  very  common  form  of 
grouping  is  one  corpuscle  surrounded  by  four  others.  This  should 
immediately  suggest  the  number  five.  In  this  way  the  counting  will 
become  more  rapid  and  also  more  reliable. 

The  calculation  is  very  simple.  The  number  of  corpuscles  in  80 
squares  di\dded  by  100  will  give  the  number  of  millions  per  cubic  milli- 
meter. If,  for  example,  536  corpuscles  have  been  counted  in  80  squares 
then  with  a  dilution  of  i :  200  the  number  of  corpuscles  per  cubic  milli- 

meter  is  5,360,000.     Thus,  ■^^^— X4,oooX2oo  =5,360,000  erythrocytes  per 

80 

cubic  millimeter.     More  than  two  decimal  places  are  without  significance. 


CHAPTER  XIII. 

MILK. 

Milk  is  the  most  satisfactory  individual  food  material  elaborated  by 
nature.  It  contains  the  three  nutrients,  protein,  fat,  and  carbohydrate 
and  inorganic  salts  in  such  proportion  as  to  render  it  a  very  acceptable 
dietary  constituent.  It  is  a  specific  product  of  the  secretory  activity  of  the 
mammary  gland.  It  contains,  as  the  principal  solids,  olein,  palmitin, 
stearin,  butyrin,  caseinogen,  lact-alhumin,  laclo- globulin,  lactose,  and  calcium 
phosphate.  It  also  contains  at  least  traces  of  lecithin,  cholesterol,  urea, 
creatine,  creatinine,  and  the  tri-glycerides  of  caproic,  lauric,  and  myristic 
acids.  Citric  acid  is  also  said  to  be  present  in  milk  in  minute  quantity. 
Considered  from  the  standpoint  of  colloid  chemistry  we  may  classify  the 
main  constituents  of  milk  as  follows:^ 

In  suspension  Fat  (olein,  palmitin,  etc.). 

r  Caseinogen — an     unstable     or     irreversible 
In    colloidal    solution  colloid. 

[  Lact-alhumin — a  stable  or  reversible  colloid. 

^  ^  11  -1      1  X-  [  Salts    (calcium  phosphate,  etc.). 

In  crystalloid  solution        I   ^         ;,  t-      t         >        j 

[  Sugar  (lactose). 

Fresh  milk  is  amphoteric  in  reaction  to  litmus,'  but  upon  standing  for 
a  sufficiently  long  time,  unsterilized,  it  becomes  acid  in  reaction,  due  to  the 
production  of  fermentation  lactic  acid, 

H     OH 

I        I 
H-C-C-COOH, 

H     H 

from  the  lactose  contained  in  it.  This  is  brought  about  through  bacterial 
activity.  The  white  color  is  imparted  to  the  milk  partly  through  the 
fine  emulsion  of  the  fat  and  partly  through  the  medium  of  the  caseinogen 
in  solution.  The  specific  gravity  of  milk  varies  somewhat,  the  average 
being  about  1.030.     Its  freezing-point  is  about  — 0.56°  C. 

Fresh  milk  does  not  coagulate  on  being  boiled  but  a  film  consisting 
of  a  combination  of  caseinogen  forms  on  the  surface.     If  the  film  be 

'  Alexander  and  Bullowa:   Jour.  Am.  Med.  Ass'n.,  55,  1196,  igio. 

-  Human  milk  as  well  as  cow's  milk.     It  is,  however,  acid  to  phenolphthalein. 

235 


236  PHYSIOLOGICAL    CHEMISTRY. 

removed,  thus  allowing  a  fresh  surface  to  come  in  contact  with  the 
air,  a  new  film  will  form  indefinitely  upon  the  application  of  heat. 
Surface  evaporation  and  the  presence  of  fat  facilitate  the  formation 
of  the  film,  but  are  not  essential  (Rettger\).  As  Jamison  and  Hertz- 
have  shown,  a  similar  film  will  form  on  heating  any  protein  solution  con- 
taining fat  or  parafiin.  If  the  milk  is  acid  in  reaction,  through  the  inception 
of  lactic  acid  fermentation,  or  from  any  other  cause,  no  film  will  form  when 
heat  is  applied,  but  instead  a  true  coagulation  will  occur.  When  milk  is 
boiled  certain  changes  occur  in  its  odor  and  taste.  These  changes, 
according  to  Rettger,^  are  due  to  a  partial  decomposition  of  the  milk 
proteins  and  are  accompanied  by  the  liberation  of  a  volatile  sulphide, 
probably  hydrogen  sulphide. 


Fig.  7g. — Normal  Milk  and  Colostrum. 
a,  Normal  milk;  h,  Colostrum. 

The  milk-curdhng  enzymes  of  the  gastric  and  the  pancreatic  juice 
have  the  power  of  splitting  the  caseinogen  of  the  milk,  through  a  process  of 
hydrolysis,  into  soluble  casein  and  a  peptone-like  body.  This  soluble 
casein  then  forms  a  combination  with  the  calcium  of  the  milk  and  an 
insoluble  curd  of  calcium  casein  or  casein  results.  The  clear  fluid  sur- 
rounding the  curd  is  known  as  whey. 

There  is  still  considerable  confusion  of  terms  when  different  authorities 
discuss  milk  proteins  and  the  actibn  of  milk  curdling  enzymes  upon  them. 
The  English-speaking  scientists  ^uite  uniformly  accept  the  classification 
of  Halliburton''  as  given  above.  On  the  other  hand,  the  Germans  in 
particular  give  the  name  casein  to  the  milk  protein  and  paracasein  to  the 

'  Rettger:  American  Journal  of  Physiolof^y,  7,  325,  1902. 
^  JamLson  and  Hertz:  Journal  of  Fhysiology,  27,  26,  igo2. 
'  Rettger:    American  Journal  oj  Physiology,  6,  450,  1902. 
'Halliburton:    Journal  of  Physiology,  11,  448,  1900. 


MILK.  237 

product  of  the  action  of  rennin  upon  this  protein.     The  confusion  of 
terms  may  be  represented  thus: 

English.  German. 

Caseinogen.  =  Casein. 

Casein.  •    =  Paracasein. 

The  most  ])ronounced  difference  between  human  milk  and  cow's  milk 
is  in  the  proteintontent,  although  there  are  also  differences  in  the  fats  and 
likewise  striking  biological  differences  difficult  to  define  chemically.  It 
has  been  shown  that  the  caseinogen  of  human  milk  differs  from  the 
caseinogen  of  cow's  milk  in  being  more  difficult  to  precipitate  by  acid  or 
coagulate  by  gastric  rennin.  The  casein  curd  also  forms  in  a  much  looser 
and  more  flocculent  manner  than  that  from  cow's  milk  and  is  for  this 
reason  much  more  easily  digested  than  the  latter.  Interesting  data 
relative  to  the  composition  of  milk  from  various  sources  may  be  gathered 
from  the  following  table  which  was  compiled  mainly  from  the  results  of 
investigations  by  Proscher^  and  by  Abderhalden-  in  Bunge's  laboratory. 
It  will  be  noted  that  the  composition  of  the  milk  varies  directly  with  the 
length  of  time  needed  for  the  young  of  the  particular  species  to  double 
in  weight. 


Period  in  which 
„       .  Weight  of  the 

Species.  New-born  is 


100  Parts  of  Milk  Contain 


Doubled  (Days).        Proteins.  Salts.         Calcium.        ^^'Jj;^''"'' 


Man '  180  1.6  0.2  0033  0047 

Horse 60  2.0  0.4  0.124  0131 

Cow 47  3.5  0.7  0.160  0.197 

Goat 22  3.7  0.8  o  197  0.284 

Sheep 15  4.9  0.8  0.245  0.293 

Pig 14  5.2  0.8  0.249  0.308 

Cat 9.5  7.0  i.o  

"Dog.. 9  7.4  1.3  0.455  0.508 

Rabbit 6  10.4  2.5  0.891  0.997 


The  secretion  of  the  mammary  glands  of  the  newborn  of  both  sexes 
is  called  '"witches'  milk."  The  name  is  centuries  old  and  evidently 
refers  to  the  mystery  of  the  useless  secretion.  Basch^  has  recently  sug- 
gested that  this  secretion  of  "wtches'  milk"  is  brought  about  by  the 
passage  of  hormones  (see  chapter  on  Pancreatic  Digestion)  from  the  blood 
of  the  mother  to  the  fetus. 

Lactose,  the  principal  carbohydrate  constituent  of  milk,  is  an  impor- 

'  Proscher:    Zeit.f.  physiol.  Chemie,  24,  285,  1898. 

^  .\bderhalden:    Ibid..  26,  487,  1899;  ^"^1  27,  pp.  408  and  457,  1899. 

*  Basch:    Miinch.  med.  Woch.,  58,  2266,  191 1. 


238  PHYSIOLOGICAL    CHEMISTRY. 

tant  member  of  the  disaccharide  group.  It  occurs  only  in  milk,  except  as 
it  is  found  in  the  urine  of  women  during  pregnancy,  during  the  nursing 
period,  and  soon  after  weaning;  it  also  occurs  in  the  urine  of  normal  per- 
sons after  the  ingestion  of  a  very  large  amount  of  lactose  in  the  food. 
It  is  not  derived  directly  from  the  blood,  but  is  a  specific  product  of  the 
cellular  activity  of  the  mammary  gland.  It  has  strong  reducing  power, 
is  dextro-rotatory,  and  forms  an  osazone  with  phenylhydrazine.  The 
souring  of  milk  is  due  to  the  formation  of  lactic  acid  from  lactose  through 
the  agency  of  the  bacterium  lactis.  Putrefactive  bacteria  in  the  alimentary 
canal  may  bring  about  this  same  reaction.  Lactose  is  not  fermentable 
by  pure  yeast.  It  was  recently  claimed  that  lactosin,  a  new  carbohy- 
drate, had  been  isolated  from  milk. 


Fig.  80. — Lactose. 

Caseinogen,  the  principal  protein  constituent  of  milk,  belongs  to  the 
group  of  phosphoproteins.  It  has  acidic  properties  and  combines  with 
bases  to  produce  salts.  It  is  not  coagulable  upon  boihng  and  is  precipi- 
tated from  its  neutral  solution  by  certain  metallic  salts  as  well  as  upon 
saturation  with  sodium  chloride  or  magnesium  sulphate.  Its  acid  solu- 
tion is  precipitated  by  an  excess  of  mineral  acid. 

Lactalbumin  and  lacto-globulin,  the  protein  constituents  of  milk, 
next  in  importance  to  caseinogen,  closely  resemble  serum  albumin  and 
serum  globulin  in  their  general  properties.  According  to  Wroblewski, 
a  protein  called  opalisin  is  also  present  in  milk. 

Butter  (milk  fat)  consists  in  large  part  of  olein  and  palmitin.  Stearin, 
butyrin,  caproin  and  traces  of  other  fats  arc  also  present.  When  butter 
becomes  rancid  through  the  cleavage  of  certain  of  its  constituent  fats  by 
bacteria  the  odors  of  caproic  and  butyric  acids  are  in  evidence. 

Colostrum  is  the  name  given  to  the  product  of  the  mammary  gland 


MILK.  239 

secreted  for  a  short  time  before  parturition  and  during  the  early  period 
of  lactation  (see  Fig.  79,  p.  236).  It  is  yellowish  in  color,  contains  more 
solid  matter  than  ordinary  milk,  and  has  a  higher  specific  granty  (1.040- 
1.080).  The  most  striking  dilTerence  between  colostrum  and  ordinary 
milk  is  the  high  percentage  of  lactalbumin  and  lacto-globulin  in  the 
former.  This  abnormality  in  the  protein  content  is  responsible  for  the 
coagulation  of  colostrum  upon  boiling. 

Such  enzymes  as  Hpase,  amylase,  galactase,  catalase,  oxidases,  peroxi- 
dases, and  reductases  have  been  identified  in  milk,  but  not  all  of  them 
in  milk  of  the  same  species  of  animal. 

Among  the  principal  preservatives  used  in  connection  with  milk  are 
formaldehyde,  hydrogen  peroxide,  boric  acid,  borates,  salicylic  acid, 
and  salicylates. 

Experiments  on  Milk. 

1.  Reaction. — Test  the  reaction  of  fresh  cow's  milk  to  litmus, 
phenol phtJialcin  and  co7igo  red. 

2.  Biuret  Test. — Make  the  biuret  test  according  to  directions  given 
on  page  98. 

3.  Microscopical  Examination. — Examine  fresh  whole  milk, 
skimmed  or  centrifugated  milk,  and  colostrum  under  the  microscope. 
Compare  the  microscopical  appearance  with  Fig.  79,  page  236. 

4.  Specific  Gravity. — Determine  the  specific  gravity  of  both  whole 
and  skimmed  milk  (see  p.  278).  Which  possesses  the  higher  specific 
gravity  ?     Explain  why  this  is  so. 

5.  Film  Formation. — Place  10  c.c.  of  milk  in  a  small  beaker  and 
boil  a  few  minutes.  Note  the  formation  of  a  film.  Remove  the  film  and 
heat  again.  Does  the  film  now  form?  Of  what  substance  is  this  film 
composed  ?  The  biuret  test  was  positive,  why  do  we  not  get  a  coagu- 
lation here  when  we  heat  to  boiling? 

6.  Coagulation  Test. — Place  about  5  c.c.  of  milk  in  a  test-tube, 
acidify  slightly  with  dilute  acetic  acid  and  heat  to  boiling.  Do  you  get 
any  coagulation  ?     Why  ? 

7.  Action  of  Hot  Alkali.— To  a  little  milk  in  a  test-tube  add  a  few 
drops  of  potassium  hydroxide  and  heat.  A  yellow  color  develops  and 
gradually  deepens  into  a  brown.  To  what  is  the  formation  of  this  color 
due? 

8.  Test  for  Chlorides.— To  about  5  c.c.  of  milk  in  a  test-tube  add 
a  few  drops  of  very  dilute  nitric  acid  to  form  a  precipitate.  Filter  off  this 
precipitate  and  test  the  filtrate  for  chlorides.  Does  milk  contain  any 
chlorides  ? 


240  PHYSIOLOGICAL    CHEMISTRY. 

9.  Guaiac  Test. — To  about  5  c.c.  of  water  in  a  test-tube  add  3  drops 
of  milk  and  enough  alcoholic  solution  of  guaiac  (strength  about  1:60)^ 
to  cause  a  turbidity.  Thoroughly  mix  the  fluids  by  shaking  and  observe 
any  change  which  may  gradually  take  place,  in  the  color  of  the  mixture. 
If  no  blue  color  appears  in  a  short  time,  heat  the  tube  gently  below  60°  C. 
and  observe  whether  the  color  reaction  is  hastened.  In  case  a  blue 
color  does  not  appear  in  the  course  of  a  few  minutes,  add  hydrogen  perox- 
ide or  old  turpentine,  drop  by  drop,  until  the  color  is  observed.  Fresh 
milk  will  frequently  give  this  blue  color  when  treated  with  an  alcoholic 
solution  of  guaiac  without  the  addition  of  hydrogen  peroxide  or  old  tur- 
pentine.    See  discussion  on  page  204. 

10.  Tests  to  Differentiate  Between  Raw  Milk  and  Heated  Milk.— 
(a)  Kastle's  Peroxidase  Reaction. — The  peroxidase  reaction  of  milk  is 
founded  upon  the  fact  that  small  amounts  of  raw  milk  will  induce  the 
oxidation  of  various  leuco  compounds  by  hydrogen  peroxide.  This 
reaction  has  been  used  in  a  practical  way  as  the  most  convenient  means 
of  differentiating  between  raw  milk  and  heated  milk.  Many  substances 
have  been  employed  for  this  purpose,  e.  g.,  guaiac,  paraphenylenediamine, 
ortol,  amidol,  etc.  Kastle  has  found  that  a  dilute  solution  of  "trikresol"^ 
acts  as  a  sensitizing  agent  in  the  peroxidase  reaction  and  offers  the  follow- 
ing test  which  is  based  upon  this  fact:  To  2-5  c.c.  of  raw  milk  in  a  test- 
tube  add  0.1-0.3  c-c.  of  M/io  hydrogen  peroxide  and  i  c.c.  of  a  i  per  cent 
solution  of  "trikresol."  A  slight  though  unmistakable  yellow  color  will 
be  observed  to  develop  throughout  the  solution. 

Repeat  the  test  using  milk  which  has  been  boiled  or  heated  to  80°  C. 
for  10-20  minutes,  and  cooled,  and  note  that  no  yellow  color  is  produced. 

The  color  reaction  in  the  case  of  the  raw  milk  probably  results  from 
the  oxidation  of  the  cresols  by  the  hydrogen  peroxide.  The  first  product 
of  this  oxidation^  then  oxidizes  the  leuco  compound,  when  such  is  present, 
and  causes  the  color  observed. 

(b)  Wilkinson  and  Peters'  Test.* — To  10  c.c.  of  the  milk  to  be  tested 
add  2  c.c.  of  a  4  per  cent  alcoholic  solution  of  benzidine,  sufficient  acetic 
acid  to  coagulate  the  milk  (usually  2-3  drops)  and  finally  2  c.c.  of  a  3  per 
cent  solution  of  hydrogen  peroxide.  Raw  milk  yields  an  immediate  blue 
color.  In  adding  the  peroxide  it  is  best  to  permit  it  to  flow  slowly  down 
the  wall  of  the  vessel  containing  the  mixture  instead  of  allowing  it  to  mix 
with  the  milk.  Milk  which  has  been  heated  to  78°  C.  or  above  remains 
unchanged. 

'  Buckmaster  advises  the  use  of  an  akoholic  solution  of  guaiaconic  acid  instead  of  an 
alcoholic  solution  of  guaiac  resin.     Guaiaconic  acid  is  a  constituent  of  guaiac  resin. 

^  "Trikresol"  is  the  trade  name  of  an  antiseptic  which  contains  the  three  cresols  in  ap- 
proximately equal  proportions. 

"  Probably  some  organic  peroxide  or  quinoid  compound. 

*  Wilkinson  and  Peters:   Z.  Nahr-Cenussm.,  16,  No.  3,  p.  172. 


MILK.  241 

11.  Saturation  with  Magnesium  Sulphate. — Place  about  5  c.c. 
of  milk  in  a  test-tube  and  saturate  with  solid  magnesium  sulphate. 
WTiat  is  this  ])recipitate  ? 

12.  Influence  of  Gastric  Rennin  on  Milk. — Prepare  a  series  of  five 
tubes  as  follows: 

(a)  5  c.c.  of  fresh  milk+0.2  per  cent  HCl  (add  drop  by  drop  until  a 
precipitate  forms). 

(b)  5  c.c.  of  fresh  milk+  5  drops  of  rennin  solution. 

(f)   5  c.c.  of  fresh  milk+  10  drops  of  0.5  per  cent  XajCOg. 

(d)  5  c.c.  of  fresh  milk-i-  10  drops  of  ammonium  oxalate. 

(e)  5  c.c.  of  fresh  milk -f  5  drops  of  0.2  per  cent  HCl. 

Now  to  each  of  the  tubes  (c),  (d)  and  (e)  add  5  drops  of  rennin  solution. 
Place  the  whole  series  of  five  tubes  at  40°  C.  and  after  10-15  minutes  note 
what  is  occurring  in  the  different  tubes.  Give  a  reason  for  each  particular 
result. 

13.  Preparation  of  Caseinogen. — Fill  a  large  beaker  one-third 
full  of  skimmed  (or  ccntrifugated)  milk  and  dilute  it  with  an  equal  volume 
of  water.  Add  dilute  hydrochloric  acid  until  a  flocculent  precipitate 
forms.  Stir  after  each  acidification  and  do  not  add  an  excess  of  the  acid 
as  the  precipitate  would  dissolve.  Allow  the  precipitate  to  settle,  decant 
the  supernatant  fluid,  and  reserve  it  for  use  in  later  (14-16)  experiments. 
Filter  off  the  precipitate  of  caseinogen  and  remove  the  excess  of  moisture 
by  pressing  it  between  filter  papers.  Transfer  the  caseinogen  to  a  small 
beaker,  add  enough  95  per  cent  alcohol  to  cover  it  and  stir  for  a  few 
moments.  Filter,  and  press  the  precipitate  between  filter  papers  to  re- 
move the  alcohol.  Transfer  the  caseinogen  again  to  a  small  dry  beaker, 
cover  the  precipitate  with  ether  and  heat  on  a  water-bath  for  ten  minutes, 
stirring  continuously.  Filter  (reserve  the  filtrate),  and  press  the  precipi- 
tate as  dry  as  possible  between  filter  papers.  Open  the  papers  and 
allow  the  ether  to  evaporate  spontaneously.  Grind  the  precipitate  to  a 
powder  in  a  mortar.  Upon  the  caseinogen  prepared  in  this  way  make  the 
following  tests: 

(a)  Solubility. — Try  the  solubility  in  the  ordinary  solvents. 
(6)   Millans  Reaction. — Make  the  test  according  to   the   directions 
given  on  page  97. 

(c)  Biuret  Test. — Make  the  test  according  to  directions  given  on 
page  98. 

((/)  Hopkins-Cole  Reaction. — Make  the  test  according  to  the  directions 
given  on  page  98. 

{e)  Loosely  Combined  Sulphur. — Test  for  loosely  combined  sulphur 
according  to  the  directions  given  on  page  108. 


16 


242  PHYSIOLOGICAL    CHEMISTRY. 

(/)  Fusion  Test  for  Phosphorus. — Test  for  phosphorus  by  fusion 
according  to  directions  given  on  page  271. 

14.  Coagulable  Proteins  of  Milk. — Place  the  filtrate  from  the 
original  caseinogen  precipitate  in  a  casserole  and  heat,  on  a  wire  gauze, 
over  a  free  flame.  As  the  solution  concentrates,  a  coagulum  consisting  of 
lactalhimin  and  lactoglobidin  will  form.  Continue  to  concentrate  the  solu- 
tion until  the  volume  is  about  one-half  that  of  the  original  solution. 
Filter  off  the  coagulable  proteins  (reserve  the  filtrate)  and  test  them  as 
follows : 

(a)  Millon^s  Reaction.— Make  the  test  according  to  the  directions  given 
on  page  97. 

(&)  Biuret  Test. — Make  the  test  according  to  the  directions  given  on 
page  98. 

(c)  Hopkins-Cole  Reaction.' — Make  the  test  according  to  the  directions 
given  on  page  98. 

15.  Detection  of  Calcium  Phosphate. — Evaporate  the  filtrate  from 
the  coagulable  proteins,  on  a  water-bath,  until  crystals  begin  to  form. 

It  may  be  necessary  to  concentrate  to  15  c.c. 
before  any  crystallization  will  be  observed. 
Cool  the  solution,  filter  off  the  crystals  (reserve 
the  filtrate),  and  test  them  as  follows: 

(a)  Microscopical    Examination.- — Examine 

the  crystals  and  compare  them  with  those  in 

Fig.  81. 
Fig.  81. — Calcium  Phosphate.  ,,,   ,-..       ,        .  ....         .  ,       ^r^     , 

[b)  Dissolve  the  crystals  m  nitric  acid.      1  est 

part  of  the  acid  solution  for  phosphates.  Render  the  remainder  of  the 
solution  slightly  alkaline  with  ammonia,  then  acidify  with  acetic  acid 
and  add  ammonium  oxalate.  Examine  the  crystals  under  the  microscope 
and  compare  them  with  those  in  Fig.  104,  p.  363. 

16.  Detection  of  Lactose. — Concentrate  the  filtrate  from  the  cal- 
cium phosphate  until  it  is  of  a  syrup-like  consistency.  Allow  it  to  stand 
over  night  and  observe  the  formation  of  crystals  of  lactose.  Make  the 
following  experiments. 

(a)  Microscopical  Examination. — Examine  the  crystals  and  com- 
pare them  with  those  in  Fig.  80,  page  238. 

(b)  Fehling's    Test. — Try  Fehling's   test   upon   the   mother   liquor. 

(c)  Phenylhydrazine  Test. — Apply  the  phenlhydrazine  test  to  some  of 
the  mother  liquor  according  to  the  directions  given  on  page  28. 

17.  Milk  Fat. — (a)  Evaporate  the  ether  filtrate  from  the  caseinogen 
(Experiment  13)  and  observe  the  fatty  residue.  The  milk  fat  was 
carried  down  with  the  precipitate  of  caseinogen  and  was  removed  when 
the  latter  was  treated  with  ether.     If  ccntrifugated  milk  was  used  in  the 


MILK.  243 

preparation  of  the  caseinogen  the  amount  of  fat  in  the  ether  filtrate  may 
be  very  small.  To  secure  a  larger  yield  of  fat  proceed  according  to  direc- 
tions given  under  (b)  below. 

{b)  To  25  c.c.  of  whole  milk  in  an  evaporating  dish  add  a  little  sand 
or  filter  paper  and  evaporate  the  fluid  to  dryness  on  a  water-bath.  Grind 
or  break  up  the  residue  after  cooling  and  extract  with  ether  in  a  flask. 
Filter  and  remove  the  ether  from  the  filtrate  by  evaporation.  How  can 
you  identify  fats  in  the  ethereal  residue  ? 

18.  Saponification  of  Butter. — Dissolve  a  small  amount  of  butter  in 
alcohol  made  strongly  alkaline  with  potassium  hydroxide.  Place  the 
alcoholic-potash  solution  in  a  casserole,  add  about  100  c.c.  of  water  and 
boil  for  10-15  minutes  or  until  the  odor  of  alcohol  cannot  be  detected. 
Place  the  casserole  in  a  hood  and  neutralize  the  solution  with  sulphuric 
acid.  Note  the  odor  of  volatile  fatty  acids,  particularly  butyric  acid. 
Under  certain  conditions  the  odor  of  ethyl  butyrate  may  also  be  detected. 

19.  Detection  of  Preservatives. — {a)  Formaldehyde. 

I.  Gallic  Acid  Test. — Acidify  30  c.c.  of  milk  with  2  c.c.  of  normal 
sulphuric  acid  and  distil.  Add  0.2-0.3  c.c.  of  a  saturated  alcoholic  solu- 
tion of  gallic  acid  to  the  first  5  c.c.  of  the  distillate,  then  incline  the  test- 
tube  and  slowly  introduce  3-5  c.c.  of  concentrated  sulphuric  acid,  allowing 
it  to  run  slowly  down  the  side  of  the  tube.  A  green  ring,  which  fmally 
changes  to  blue,  is  formed  at  the  juncture  of  the  fluids.  This  is  claimed, 
by  Sherman,  to  be  twice  as  delicate  as  either  the  sulphuric  acid  or  the 
hydrochloric  acid  test  for  formaldehyde. 

II.  Leaches  Hydrochloric  Acid  Test. — Mix  10  c.c.  of  milk  and  10  c.c. 
of  concentrated  hydrochloric  acid  containing  about  0.002  gram  of  ferric 
chloride  in  a  small  porcelain  evaporating  dish  or  casserole  and  gradually 
raise  the  temperature  of  the  mixture,  on  a  water-bath,  nearly  to  the 
boiling-point,  w^ith  occasional  stirring.  If  formaldehyde  is  present  a 
violet  color  is  produced,  while  a  brown  color  develops  in  the  absence  of 
formaldehyde.  In  case  of  doubt  the  mixture,  after  having  been  heated 
nearly  to  the  boiling-point  for  about  one  minute,  should  be  diluted  with 
50-75  c.c.  of  water,  and  the  color  of  the  diluted  fluid  carefully  noted,  since 
the  violet  color  if  present  will  quickly  disappear.  Formaldehyde  may  be 
detected  by  this  test  when  present  in  the  proportion  i :  250,000. 

(b)  Salicylic  and  Salicylates. — Remont's  Method.'  Acidify  20  c.c.  of 
milk  with  sulphuric  acid,  shake  well  to  break  up  the  curd,  add  25  c.c.  of 
ether,  mix  thoroughly,  and  allow  the  mixture  to  stand.  By  means  of  a 
pipette  remove  5  c.c.  of  the  ethereal  extract,  evaporate  it  to  dryness,  boil 
the  residue  with  10  c.c.  of  40  per  cent  alcohol,  and  cool  the  alcoholic 
solution.     Make  the  volume  10  c.c,  filter  through  a  dry  paper  if  necessary 

'  Sherman's  Organic  Analysis,  First  Edition,  p.  232. 


244  PHYSIOLOGICAL    CHEMISTRY. 

to  remove  fat,  and  to  5  c.c.  of  the  filtrate,  which  represents  2  c.c.  of  milk, 
add  2  c.c.  of  a  2  per  cent  solution  of  ferric  chloride.  The  production  of  a 
purple  or  violet  color  indicates  the  presence  of  salicylic  acid. 

This  test  may  form  the  basis  of  a  quantitative  method  by  diluting  the 
final  solution  to  50  c.c.  and  comparing  this  with  standard  solutions  of 
salicylic  acid.  The  colorimetric  comparisons  maybe  made  in  a  Duboscq 
colorimeter. 

(f)  Hydrogen  Peroxide. — Add  2-3  drops  of  a  2  per  cent  aqueous 
solution  of  para-phenylenediamine  hydrochloride  to  10-15  c.c.  of  milk. 
If  hydrogen  peroxide  is  present  a  blue  color  will  be  produced  immediately 
upon  shaking  the  mixture  or  after  allowing  it  to  stand  for  a  few  minutes. 
It  is  claimed  that  hydrogen  peroxide  may  be  detected  by  this  test  when 
present  in  the  proportion  1:40,000. 

{d)  Boric  Acid  and  Borates. — To  the  ash,  obtained  according  to  the 
directions  given  in  Experiment  4,  page  438,  add  2  drops  of  dilute  hydro- 
chloric acid  and  i  c.c.  of  water.  Place  a  strip  of  turmeric  paper  in  the 
dish  and  after  allowing  it  to  soak  for  about  one  minute  remove  it  and  allow 
it  to  dry  in  the  air.  The  presence  of  boric  acid  is  indicated  by  the  pro- 
duction of  a  deep  red  color  which  changes  to  green  or  blue  upon  treatment 
with  a  dilute  alkali.  This  test  is  supposed  to  show  boric  acid  when 
present  in  the  proportion  1:8000. 


CHAPTER  XIV. 


Epithelial  and  connective  tissues, 
epithelial  tissue  (keratin). 

The  albuminoid  keratin  constitutes  the  major  portion  of  hair,  horn, 
hoof,  feathers,  nails,  and  the  epidermal  layer  of  the  skin.  There  is  a 
group  of  keratins  the  members  of  which  possess  very  similar  properties. 
The  keratins  as  a  group  are  insoluble  in  the  usual  protein  solvents  and 
are  not  acted  upon  by  the  gastric  or  pancreatic  juices.  They  all  respond 
to  the  xanthoproteic  and  Millon  reactions  and  are  characterized  by  con- 
taining large  amounts  of  sulphur.  Keratin  from  any  of  its  sources  may 
be  prepared  in  a  pure  form  by  treatment,  in  sequence,  with  artificial 
gastric  juice,  artificial  pancreatic  juice,  boiling  alcohol,  and  boiling  ether, 
from  twenty-four  to  forty-eight  hours  being  devoted  to  each  process. 

The  percentage  composition  of  some  typical  keratins  is  given  in  the 
following  table: 


Percentage  Composition. 

S 

N                      C                      H 

1 

0 

Nails' 

2.80 

17.51        '        5100                 6.94 

1 

21.75 

.  Horn- 

1 

3.20 

';o  86 

6.94 

S 

Indian 

4.82 

15.40               44.06                 6.53 

29.19 

Japanese. . .  . 
Negro 

4.96 

14.64               42.99                  5-91 

31-50 

4.84 

14.90               43.85         1         6.37 

30.04 

Caucasian 
(adults). 

5.22 

1        15.79                44-49                  6.44 

28.66 

Caucasian 
(children) . 

'         4-93 

1       1458                43  23 

6.46 

30.80 

The  composition  of  human  hair  is  influenced  by  its  color  and  by  the 
race,  sex,  age  and  purity  of  breeding  of  the  individual.^ 

'  Mulder:  Versuch  einer  allgent.  physiol.  Chem.,  Braunschweig,  1844-51. 
-  Horhaczewski:  Ladenburg^ s  Handworterbuch  d.  Chem.,  3. 
'  Rutherford  and  Hawk:  Jour.  Biol.  Chem.,  3,  459,  1907. 

245 


246  physiological  chemistry. 

Experiments  on  Epithelial  Tissue. 

Keratin. 

Horn  shavings  or  nail  parings  may  be  used  in  the  experiments  which 
follow: 

1.  Solubility. — Test  the  solubility  of  keratin  in  the  ordinary  solvents 
(see  page  27). 

2.  Afillon^s  Reaction. 

3.  Xanthoproteic  Reaction. 

4.  Adamkiewicz^ s  Reaction. 

5.  Hopkins-Cole  Reaction. 

6.  Test  for  Loosely  Combined  Sulphur. 


CONNECTIVE  TISSUE. 
I.  WHITE  FIBROUS  TISSUE. 

The  principal  solid  constituent  of  white  fibrous  connective  tissue  is  the 
albuminoid  collagen.  This  body  is  also  found  in  smaller  percentage  in 
cartilage,  bone,  and  ligament,  but  the  callogen  from  the  various  sources 
is  not  identical  in  composition.  In  common  with  the  keratins,  collagen 
is  insoluble  in  the  usual  protein  solvents.  It  differs  from  keratin  in  con- 
taining less  sulphur.  One  of  the  chief  characteristics  of  collagen  is, 
according  to  Hofmeister,  the  property  of  being  hydrolyzed  by  boiling  acid 
or  water  with  the  formation  of  gelatin.  Emmett  and  Gies^  claim  that 
under  these  conditions  there  is  an  intramolecular  rearrangement  of 
collagen  and  the  resultant  gelatin  is  consequently  not  the  product  of 
hydrolysis.  The  liberation  of  ammonia  from  the  collagen  during  the 
process  apparently  confirms  this  view.  Collagen  gives  Millon's  reaction 
as  well  as  the  xanthoproteic  and  biuret  tests. 

The  form  of  white  fibrous  tissue  most  satisfactory  for  general  experi- 
ments is  the  tendo  Achillis  of  the  ox.  According  to  Buerger  and  GieSj 
the  fresh  tissue  has  the  following  composition: 

Water; 62  .87% 

Solids 37-13 

Inorganic  matter o .  47 

Organic  matter 36.66 

Fatty  substance  (ether-soluble) i .  04 

Coagulable  protein 0.22 

Mucoid 1-28 

Elastin i  .63 

Collagen 31-59 

Extractives,  etc o. 90 

'  Emmett  and  Geis:  Jour.  Biol,  chem.,  3,  xxxiii  (Proceedings),  1907. 
-'  Buerger  and  Gies:  Am.  Jour.  Physiol.,  6,  219,  1901. 


EPITHELIAL  AND    CONNECTIVE    TISSUES.  247 

The  mucoid  munlioncd  above  is  called  tendomucoid^  and  is  a  glyco 
protein.   It  possesses  properties  similar  to  those  of  other  connective-tissue 
mucoids,  e.  g.,  osseomucoid  and  chondromucoid. 

Gelatin,  the  body  which  results  from  the  hydrolysis  of  collagen  (see 
statement  of  Emmett  and  Gies  above) ,  is  also  an  albuminoid.  It  responds 
to  nearly  all  the  protein  tests.  It  dififers  from  the  keratins  and  collagen  in 
being  easily  digested  and  absorbed.  Gelatin  is  not  a  satisfactory  sub- 
stitute for  the  protein  constituents  of  a  normal  diet,  however,  since  a 
certain  portion  of  its  nitrogen  is  not  available  for  the  uses  of  the  organism. 
Gelatin  from  cartilage  differs  from  gelatin  from  other  sources  in  containing 
a  lower  percentage  of  nitrogen.  Tyrosine  and  tryptophane  are  not 
numbered  among  the  decomposition  products  of  gelatin,  hence  it  does  not 
respond  to  Millon's  reaction  or  the  Hopkins-Cole  reaction. 

Experiments  on  White  Fibrous  Tissue. 

The  tendo  Achillis  of  the  ox  may  be  taken  as  a  satisfactory  type  of  the 
white  fibrous  connective  tissue. 

1.  Preparation  of  Tendomucoid. — Dissect  away  the  fascia  from 
about  the  tendon  and  cut  the  clean  tendon  into  small  pieces.  Wash  the 
pieces  in  running  w^ater,  subjecting  them  to  pressure  in  order  to  remove 
as  much  as  possible  of  the  soluble  protein  and  inorganic  salts.  This 
washing  is  very  important.  Transfer  the  washed  pieces  of  tendon  to  a 
flask  and  add  300  c.c.  of  half- saturated  lime  water.^  Shake  the  flask  at 
intervals  for  twenty-four  hours.  Filter  off  the  pieces  of  tendon  and  pre- 
cipitate the  mucoid  with  dilute  hydrochloric  acid.  Allow  the  mucoid 
precipitate  to  settle,  decant  the  supernatant  fluid  and  filter  the  remainder. 
Test  the  mucoid  as  follows : 

{a)  Solubility. — Try  the  solubility  in  the  ordinary  solvents  (see  page  27). 

(6)  Biuret  Test. — First  dissolve  the  mucoid  in  potassium  hydroxide 
solution  and  then  add  a  dilute  solution  of  copper  sulphate. 

(c)   Test  for  Loosely  Combined  Sulphur. 

{d)  Hydrolysis  of  Tendomucoid. — ^Place  the  remainder  of  the  mucoid 
in  a  small  beaker,  add  about  30  c.c.  of  water  and  2  c.c.  of  dilute  hydro- 
chloric acid  and  boil  until  the  solution  becomes  dark  brown.  Cool  the 
solution,  neutralize  it  with  concentrated  potassium  hydroxide,  and  test  by 
Fehling's  test.  With  a  reduction  of  Fehling's  solution  and  a  positive 
biuret  test  what  do  you  conclude  regarding  the  nature  of  tendomucoid  ? 

2.  Collagen. — This  substance  is  present  in  the  tendon  to  the  extent  of 
about  32  per  cent.     Therefore  in  making  the  following  tests  upon  the 

'Cutter  and  Gies:  Am.  Jour.  Physiol.,  6,155,  iQoi. 

-  Made  by  mixing  equal  volumes  of  saturated  lime  water  and  water  from  the  faucet. 


248  PHYSIOLOGICAL    CHEMISTRY. 

pieces  of  tendon  from  which  the  mucoid,  soluble  protein,  and  inorganic 
salts  were  removed  in  the  last  experiment,  we  may  consider  the  tests  as 
being  made  upon  collagen. 

(a)  Sohibility. — Cut  the  collagen  into  very  fine  pieces  and  try  its 
solubility  in  the  ordinary  solvents  (see  page  27). 

{b)  Millon's  Reaction. 

(c)  Biuret  Test. 

(d)  Xanthoproteic  Reaction, 
{e)  Hopkins-Cole  Reaction. 

(/)  Test  for  Loosely  Combined  Sulphur. — Take  a  large  piece  of  collagen 
in  a  test-tube  and  add  about  5  c.c.  of  potassium  hydroxide  solution. 
Heat  until  the  collagen  is  partly  decomposed,  then  add  1-2  drops  of  lead 
acetate  and  again  heat  to  boiling. 

(g)  Formation  of  Gelatin  from  Collagen. — Transfer  the  remainder  of  the 
pieces  of  collagen  to  a  casserole,  fill  the  vessel  about  two-thirds  full  of 
water  and  boil  for  several  hours,  adding  water  at  intervals  as  needed. 
By  this  means  the  collagen  is  transformed  and  a  body  known  as  gelatin  is 
produced  (see  p.  247). 

3.  Gelatin. — On  the  gelatin  formed  from  the  transformation  of  colla- 
gen in  the  above  experiment  (g),  or  on  gelatin  furnished  by  the  instructor 
make  the  following  tests: 

(a)  Solubility. — Try  the  solubility  in  the  ordinary  solvents  (see  page 
27)  and  in  hot  water. 

(b)  Millon's  Reaction. 

ic)  Hopkins-Cole  Reaction. — Conduct  this  test  according  to  the  modi- 
fication given  on  page  98. 

id)   Test  for  Loosely  Combined  Sulphur. 

Make  the  following  tests  upon  a  solution  of  gelatin  in  hot  water: 
{a)  Precipitation   by    Mineral    Acids. — Is    it    precipitaed    by    strong 
mineral  acids  such  as  concentrated  hydrochloric  acid  ? 

(b)  Salting-out  Experiment. — Saturate  a  little  of  the  solution  with 
solid  ammonium  sulphate.  Is  the  gelatin  precipitated  ?  Repeat  the 
experiment  with  sodium  chloride.     What  is  the  result? 

(c)  Precipitation  by  Metallic  Salts.  —Is  it  precipitated  by  metallic  ^alts 
such  as  copper  sulphate,  mercuric  chloride,  and  lead  acetate? 

{d)  Coagulation  Test. — Does  it  coagulate  upon  boiling? 

{e)  Precipitation  by  Alkaloidal  Reagents. — Is  it  precipitated  by  such 
reagents  as  picric  acid,  tannic  acid,  and  trichloracetic  acid? 

(J)  Biuret  Test. — Does  it  respond  to  the  biuret  test  ? 

ig)  Bardach's  Reaction. — Does  it  yield  the  typical  crystals  of  this 
reaction?     (See  page  10 1.)  ^ 


EPITHELIAL   AND   CONNECTIVE   TISSUES.  249 

{li)  Precipitatian  by  Alcohol. — Fill  a  test-tube  one-half  full  of  95  per 
cent  alcohol  and  pour  in  a  small  amount  of  concentrated  gelatin  solution. 
Do  you  get  a  precipitate  ?  How  would  you  prepare  pure  gelatin  from 
the  tcndo  A  chill  is  of  the  ox? 

11.  YELLOW  ELASTIC  TISSUE   (ELASTIN). 

The  ligameniitm  niichce  of  the  ox  may  be  taken  as  a  satisfactory  type  of 
the  vellow  elastic  connective  tissue.  The  principal  solid  constituent  of 
this  tissue  is  elaslin,  a  member  of  the  albuminoid  group.  In  common  with 
the  keratins  and  collagen,  elastin  is  an  insoluble  body  and  gives  the  pro- 
tein color  reactions.  It  differs  from  keratin  principally  in  the  fact  that  it 
may  be  digested  by  enzymes  and  that  it  contains  a  very  small  amount  of 
sulphur. 

It  has  recently  been  demonstrated  that  elastin  has  the  property  of 
adsorbing  pepsin  from  the  gastric  juice  and  thus  protecting  it  so  the 
enzyme  can  function  later  in  the  intestine^  (see  chapter  on  Gastric 
Digestion). 

Yellow  elastic  tissue  also  contains  mucoid  and  collagen  but  these  arc 
present  in  much  smaller  amount  than  in  white  fibrous  tissue,  as  may  be 
seen  from  the  following  percentage  composition  of  the  fresh  Ugamentiim 
nuchcB  of  the  ox  as  determined  by  Vandegrift  and  Gies." 

Water 57-57% 

Solids 42  -43 

Inorganic  matter o  •  47 

Organic  matter 41  •  96 

Fatty  substance  (ether-soluble) 1.12 

Coagulable  protein 0.62 

Mucoid ^Si 

Elastin 31-67 

Collagen 7-23 

Extractives,  etc o .  80 

Experiments  on  Elastin. 

I.  Preparation  of  Elastin  (Richards  and  Gies).^ — Cut  the  liga- 
ment into  fine  strips,  run  it  through  a  meat  chopper  and  wash  the  finely 
divided  material  in  cold,  running  water  for  24-48  hours.  Add  an  excess 
of  half-saturated  lime  water  (see  note  at  the  bottom  of  p.  247)  and 
allow  the  hashed  ligament  to  extract  for  48-72  hours.  Decant  the  lime 
water,  remove  all  traces  of  alkali  by  washing  in  water  and  then  boil  in 
water  with  repeated  renewals  until  only  traces  of  protein  material  can  be 
detected  in  the  wash  water.  Decant  the  fluid  and  boil  the  ligament  in 
10  per  cent  acetic  acid  for  a  few  hours.     Treat  the  pieces  with  5  per  cent 

'  Abderhalden  and  Meyer:  Zeit.  physiol.  Chem.,  74,  67,  191 1. 
-Vandegrift  and  Gies:  Am.  Jour.  Physiol.,  5,  287,  iqoi. 
'  Richards  and  Gies:  Am.  Jour.  Physiol.,  7,  93,  1902. 


250  PHYSIOLOGICAL   CHEMISTRY. 

hydrochloric  acid  at  room  temperature  for  a  similar  period,  extract  again 
in  hot  acetic  acid  and  in  cold  hydrochloric  acid.  Wash  out  traces  of  acid 
by  means  of  water  and  then  thoroughly  dehydrolyze  by  boiling  alcohol 
and  boiling  ether  in  turn.  Dry  in  an  air-bath  and  grind  to  a  powder  in  a 
mortar. 

2.  Solubility. — Try  the  solubility  of  the  finely  divided  elastin,  pre- 
pared by  yourself  or  furnished  by  the  instructor,  in  the  ordinary  solvents 
(see  page  27).     How  does  its  solubility  compare  with  that  of  collagen? 

3.  Millon's  Reaction. 

4.  Xanthoproteic  Reaction. 

5.  Biuret  Test. 

6.  Hopkins-Cole  Reaction. — Conduct  this  test  according  to  the 
modification  given  on  page  98. 

7.  Test  for  Loosely  Combined  Sulphur. 

III.  CARTILAGE. 

The  principal  solid  constituents  of  the  matrix  of  cartilaginous  tissue 
are  chondromucoid,  chondroitin- sulphuric  acid,  chondroalbumoid  and  collagen. 
Chondromucoid  differs  from  the  mucoids  isolated  from  other  connective 
tissues  in  the  large  amount  of  chondroitin-sulphuric  acid  obtained  upon 
decomposition.  Besides  being  an  important  constituent  of  all  forms  of 
cartilage,  chondroitin-sulphuric  acid  has  been  found  in  bone,  ligament, 
the  mucosa  of  the  pig's  stomach,  the  kidney  of  the  ox,  the  inner  coats  of 
large  arteries  and  in  human  urine.  It  may  be  decomposed  through  the 
action  of  acid  and  yields  a  nitrogenous  body  known  as  chondroitin  and 
later  this  body  yields  chondrosin.  Chondrosin  is  also  a  nitrogenous  body 
and  has  the  power  of  reducing  Fehling's  solution  more  strongly  than 
dextrose.  Sulphuric  acid  is  a  by-product  in  the  formation  of  chondroitin, 
and  acetic  acid  is  a  by-product  in  the  formation  of  chondrosin. 

Chondroalbumoid  is  similar  in  some  respects  to  elastin  and  keratin. 
It  dijGfers  from  keratin  in  being  soluble  in  gastric  juice  and  in  containing 
considerably  less  sulphur  than  any  member  of  the  keratin  group.  It 
gives  the  usual  protein  color  reactions. 

Experiments  on  Cartilage. 

1.  Preparation  of  the  Cartilage. — Boil  the  trachea  of  an  ox  in 
water  until  the  cartilage  rings  may  be  completely  freed  from  the  sur- 
rounding tissue.  Use  the  cartilage  so  obtained  in  the  following  experi- 
ments: 

2.  Solubility. — Cut  one  of  the  rings  into  very  small  pieces  and  try  the 
solubility  of  the  cartilage  in  the  ordinary  solvents  (see  page  27). 


EPITHELIAL    AND    CONNECTIVE   TISSUES.  25 1 

3.  Millon's  Reaction. 

4.  Xanthoproteic  Reaction. 

5.  Hopkins-Cole  Reaction. — Conduct  this  test  according  to  the 
modification  given  on  page  98. 

6.  Test  for  Loosely  Combined  Sulphur. 

7.  Preparation  of  Cartilage  Gelatin. — Cut  the  remaining  cartilage 
rings  into  small  pieces,  place  them  in  a  casserole  with  water  and  boil  for 
several  hours.  Filter  w^hile  the  solution  is  still  hot.  Observe  that  the 
filtrate  soon  becomes  more  or  less  solid.  What  is  the  reason  for  this? 
Bring  a  portion  of  the  material  into  solution  by  heat  and  try  the  following 
tests: 

(a)  Biuret  Test. 

(b)  Bardach's  Reaction. 

(c)  Test  for  Loosely  Combined  Sulphur. 

(d)  To  about  5  c.c.  of  the  solution  in  a  test-tube  add  a  few  drops  of 
barium  chloride.  Do  you  get  a  precipitate,  and  if  so  to  what  is  the  pre- 
cipitate due  ? 

(e)  To  about  5  c.c.  of  the  solution  in  a  test-tube  add  a  few  drops  of 
dilute  hydrochloric  acid  and  boil  for  a  few  moments.  Now  add  a  little 
barium  chloride  to  this  solution.  Is  the  precipitate  any  larger  than 
that  obtained  in  the  preceding  experiment  ?     Why  ? 

(J)  To  the  remainder  of  the  solution  add  a  little  dilute  hydrochloric 
acid  and  boil  for  a  few  moments.  Cool  the  solution,  neutralize  with 
solid  potassium  hydroxide,  and  try  Fehling's  test.     Explain  the  result. 

IV.  OSSEOUS  TISSUE. 

Of  the  solids  of  bone  about  equal  parts  are  organic  and  inorganic 
matter.  The  organic  portion,  called  ossein,  may  be  obtained  by  removing 
the  inorganic  salts  through  the  medium  of  dilute  acid.  Ossein  is  practi- 
cally the  same  body  which  is  termed  collagen  in  the  other  connective 
tissues,  and  in  common  with  collagen  yields  gelatin  upon  being  boiled 
with  dilute  mineral  acid. 

In  common  with  the  other  connective  tissues  bone  contains  a  mucoid 
and  an  albumoid.  Because  of  their  origin  these  bodies  are  called  osseo- 
mucoid and  osseoalbumoid.  Osseomucoid,  when  boiled  with  hydro- 
chloric acid,  yields  sulphuric  acid  and  a  substance  capable  of  reducing 
Fehling's  solution.  The  composition  of  osseomucoid  is  very  similar 
to  that  of  tendomucoid  and  chondromucoid  (see  page  113). 

The  inorganic  basis  of  the  dry,  fat-free  bone  is  a  chemical  substance, 
not  a  mixtur/s.  This  fact  is  indicated  by  the  uniform  composition  of 
the  bones  of  fasting  animals  as  well  as  by  the  definite  relationship  existing 


252  PHYSIOLOGICAL    CHEMISTRY. 

between  the  elements  present.  Bones  of  normal  and  fasting  animals 
of  the  same  species  present  no  profound  differences  in  percentage  compo- 
sition. The  percentage  composition  of  the  dry,  fat-free  femurs  of  two 
dogs^  after  the  animals  had  fasted  for  104  and  14  days  respectively  was 
as  follows: 


Dog.  No. 

Length  of  fast. 

Ash. 

N. 

CaO. 

MgO. 

I 

p.o^. 

I. 

104  days. 

61 .50 

4.6 

33-3 

o.S 

12.80 

2. 

14  days. 

61.56 

41 

33-^ 

O.Q 

12  .90 

The  marked  uniformity  in  composition  notwithstanding  the  wide 
variation  in  the  fasting  periods  is  significant.  The  tensile  strength  of 
the  femur  of  the  dog  has  been  found  to  be  at  least  25,000  pounds  to  the 
square  inch^  whereas  that  of  oak  is  10,000  and  that  of  cast  iron  20,000 
pounds  to  the  square  inch. 

Experiment  on  Osseous  Tissue. 

Qualitative  Analysis  of  Bone  Ash. — Take  i  gram  of  bone  ash  in 
a  small  beaker  and  add  a  little  dilute  nitric  acid.  What  does  thf  efferves- 
cence indicate  ?  Stir  throughly  and  when  the  major  portion  of  the  ash  is 
dissolved  add  an  equal  volume  of  water  and  filter.  To  the  acid  filtrate 
add  ammonium  hydroxide  to  alkaline  reaction.  A  heavy  white  pre- 
cipitate of  phosphates  results.  (What  phosphates  are  precipitated 
here  by  the  ammonia  ?)  Filter  and  test  the  filtrate  for  chlorides,  sulphates, 
phosphates,  and  calcium.  Add  dilute  acetic  acid  to  <he  precipitate  on 
the  paper  and  test  this  filtrate  for  calcium  and  phosphates.  To  the 
precipitate  remaining  undissolved  on  the  paper  add  a  little  dilute  hydro- 
chloric acid  and  test  this  last  filtrate  for  phosphates  and  iron. 

Reference  to  the  following  scheme  may  facilitate  the  analysis. 

*  Johnston  and  Hawk:  Unpublished  data.  For  data  on  a  117-day  fast  by  dog  No.  i,  see 
Howe,  Mattill  and  Hawk:  Jour.  Biol.  Chem.,  11,  103,  1912. 


EPITHELIAL   AND    CONNECTIVE    TISSUES. 
BONE  ASH. 


253 


Add  dilute  nitric  acid,  stir  thoroughly  and  after  the  major  portion  of  the  ash  has  been 
brought  into  solution  add  a  little  distilled  water  and  filter. 


Residue  I.  Filtrate  I. 

(disiard)  Add     ammonium     hydroxide     to 

alkaline  reaction  and  filter. 


Residue  II. 

Treat  oti  paper  with  acetic  acid. 


Residue  III. 

Treat  i»i  paper  with  hydro- 
chloric acid. 


Filtrate  III. 

Test  for: 

1.  Phosphates. 

2.  Calcium. 


Filtrate  II. 
Test  for: 

1.  Chlorides. 

2.  Sulphates. 

3.  Phosphates. 

4.  Calcium. 


Filtrate  IV. 
Test  for: 

1.  Iron. 

2.  Phosphates. 

y.  ADIPOSE  TISSUE. 
For  discussion  and  experiments  see  chapter  on  Fats,  page  139. 


CHAPTER  XV. 
MUSCULAR  TISSUE. 

The  muscular  tissues  are  divided  physiologically  into  the  voluntary 
(striated)  and  the  involuntary  (non-striated  or  smooth).  In  the  chemical 
examination  of  muscular  tissue  the  voluntary  form  is  generally  employed. 
Muscle  contains  about  25  per  cent  of  solid  matter,  of  which  about  four- 
fifths  is  protein  material  and  the  remaining  one-fifth  extractives  and 
inorganic  salts. 

The  proteins  are  the  most  important  of  the  constituents  of  muscular 
tissue.  In  the  living  muscle  we  find  two  proteins,  myosinogen  and  para- 
myosinogen. These  may  be  shown  to  be  present  in  muscle  plasma  ex- 
pressed from  fresh  muscles.  In  common  with  the  plasma  of  the  blood 
this  muscle  plasma  has  the  power  of  coagulating,  and  the  clot  formed  in 
this  process  is  called  myosin.  According  to  Halliburton^  and  others  in 
the  onset  of  rigor  mortis  we  have  an  indication  of  the  formation  of  this 
myosin  clot  within  the  body.  The  relation  between  the  proteins  of 
living  and  dead  muscle  is  represented  graphically  by  Halliburton  as 
follows: 

Proteins  of  the  living  muscle. 


Para-myosinogen  (25%).  Myosinogen  (75%). 

Soluble  myosin. 

Myosin. 
(The  protein  of  the  muscle  clot.) 

Of  the  total  protein  content  of  li\TLng  muscle  about  75  per  cent  is  made 
up  by  the  myosinogen  and  the  remaining  25  per  cent  is  para-myosinogen. 
These  proteins  may  be  separated  by  subjecting  the  muscle  plasma  to 
fractional  coagulation  in  the  usual  way.  Under  these  conditions  the 
para-myosinogen  is  found  to  coagulate  at  47°  C.  and  the  myosinogen  to 
coagulate  at  56°  C.  It  is  also  claimed  by  some  investigators  that  it  is 
possible  to  separate  these  two  proteins  by  the  fractional  ammonium 
sulphate  method,  but  the  possibility  of  making  an  accurate  separation  by 
this  method  is  somewhat  doubtful.  It  is  well  established  that  para- 
myosinogen is  a  globulin  since  it  responds  to  certain  of  the  protein  precip- 
itation tests  and  is  insoluble  in  water.     Myosinogen,  on  the  contrary, 

'  Halliburton:  Biochemistry  of  Muscle  and  Nerve,  1904,  p.  4. 

254 


MUSCULAR   TISSUE.  255 

is  not  a  typical  globulin  since  it  is  soluble  in  water.  It  has  been  called  a 
pseudo-globulin.  Myosin  possesses  the  globulin  characteristics.  It  is 
insoluble  in  water  but  soluble  in  the  other  protein  solvents  and  is  precipi- 
tated from  its  solution  upon  saturation  with  sodium  shloride. 

Mellanby  has  recently  reported  observations  which  he  claims  indicate 
that  there  is  only  one.  protein  in  muscle  and  that  rigor  mortis  is  due  to  the 
coagulation  of  this  protein  under  the  combined  inlluences  of  the  salt 
present  in  the  muscle  and  the  lactic  acid  developed  upon  the  death  of  the 
muscle.  He  further  states  that  the  disappearance  of  rigor  is  due  to  the 
fact  that  the  lactic  acid  which  is  continually  formed  brings  this  protein 
into  solution.  There  is  a  difference  of  opinion  as  to  whether  true  rigor 
ever  occurs  in  conection  with  non-striated  (smooth)  ^  muscle. 

Our  ideas  concerning  the  cause  of  rigor  have  undergone  an  impor- 
tant re\dsion  quite  recently.  A  very  attractive  theory  has  been  advanced 
by  Meigs^  and  experimental  confirmation  has  been  accorded  it  by  von 
Fiirth  and  Lenk.'  According  to  this  theory,  rigor  has  no  connection 
with  the  coagulation  of  the  muscle  proteins  and  may  even  be  hindered 
or  prevented  by  such  coagulation.  The  cause  of  rigor,  from  this  new 
view  point,  lies  in  the  imbibition  of  water  by  the  muscle  colloids.  It  is  well 
known  that  colloids  possess  the  property  of  absorbing  whatever  fluid 
may  be  in  contact  with  them.  Moreover,  the  capacity  of  the  colloid  for 
water  is  increased  if  the  fluid  is  slightly  acid  in  reaction.  Therefore  the 
postmortem  production  of  lactic  acid  facilitates  the  imbibition  of  muscle 
fluid  by  the  muscle  colloids.  Under  such  conditions,  the  fibers  swell, 
become  rigid  and  the  condition  known  as  rigor  mortis  results.  The 
disappearance  of  rigor  is  believed  to  be  due  to  the  coagulation  of  the 
muscle  protein  through  the  agency  of  the  accumulated  lactic  acid. 
This  change  is  accompanied  by  a  release  of  the  imbibed  water  by  the 
colloids,  inasmuch  as  the  capacity  of  a  colloid  for  retaining  fluid  is  lowered 
by  coagulation. 

Under  the  name  extractives  w^e  class  a  number  of  muscle  constituents 
which  occur  in  traces  in  the  tissue  and  may  be  extracted  by  water,  alcohol, 
or  ether.  There  are  two  classes  of  these  extractives,  the  non-nitrogenous 
extractives  and  the  nitrogenous  extractives.  Grouped  under  the  non- 
nitrogenous  bodies  we  have  glycogen,  dextrin,  sugars,  lactic  acid,  inosite, 
C5Hg(OH)g,  and  fat.  In  the  class  of  nitrogenous  extractives  we  have 
creatine,  creatinine,  xanthine,  hypoxanthine,  uric  acid,  urea,  carnine,  guanine, 
phosphocarnic  acid,  inosinic  acid,  carnosine,  taurine,  carnitine,  novaine, 
ignotine,  neosine,  oblitine,  carnomiiscarine  and  methylguanidine  (see  for- 
mulas on  page  260).     Not  all  of  these  extractives  are  present  in  the 

'  Saxl:  Beitrdge  zur  chcmischeti  Physiologic  und  Pathologic,  g,  i,  IQ07. 

^  Meigs:  American  Journal  of  Physiology,  26,  191,  igio. 

'  von  Fiirth  and  Lenk:  Wiener  klinische  Wochenschrift,  24,  1079,  igri. 


25O  PHYSIOLOGICAL    CHEMISTRY. 

muscles  of  all  species  of  animals.  Other  extractives  besides  those 
enumerated  above  have  been  described  and  there  are  undoubtedly  still 
others  whose  presence  remains  undetermined.  A  detailed  consideration 
would,  however,  be  unprofitable  in  this  place. 

Glycogen  is  an  important  constituent  of  muscle.  The  content 
of  this  polysaccharide  in  muscle  varies  and  is  markedly  decreased  by 
intense  muscular  activity.  It  is  transformed  into  sugar  and  used  as 
fuel.  The  liver  is  the  organ  which  stores  the  reserve  supply  of  glycogen 
and  transforms  it  into  dextrose  w^hich  is  passed  into  the  blood  stream  and 
so  carried  to  the  working  muscle  where  it  is  synthesized  into  glycogen. 
The  glycogen  thus  formed  is  then  changed  into  dextrose  as  the  working 
muscle  may  need  it. 

Glycogen  is  a  polysaccharide  and  has  the  same  percentage  com- 
position as  starch  and  dextrin.  It  resembles  starch  in  forming  an  opal- 
escent solution  and  resembles  dextrin  in  being  very  soluble,  in  giving  a 
reddish  color  with  iodine  and  in  being  dextro-rotatory.  Glycogen  may 
be  prepared  from  muscle  by  extracting  with  boiling  water  and  then 
precipitating  the  glycogen  from  the  aqueous  solution  by  alcohol:  dilute 
or  concentrated  potassium  hydroxide  may  also  be  used  to  extract  the 
glycogen.  Glycogen  may  be  prepared  in  the  form  of  a  white,  tasteless, 
amorphous  powder.  It  is  completely  precipitated  from  its  solution  by 
saturation  with  solid  ammonium  sulphate,  but  is  not  precipitated  by 
saturation  with  sodium  chloride.  It  may  also  be  precipitated  by  alcohol, 
tannic  acid,  or  ammoniacal  basic  lead  acetate.  It  has  the  power  of 
holding  cupric  hydroxide  in  solution  in  alkaline  fluids  but  cannot  reduce 
it.  It  may  be  hydrolyzed  with  the  formation  of  dextrose  by  dilute  mineral 
acids  and  is  readily  digested  by  amylolytic  enzymes. 

Mendel  and  Leavenworth  have  recently  drawn  the  conclusion, 
from  the  examination  of  embryo  pigs,  that  embryonic  structures  do  not 
contain  exceptionally  large  amounts  of  glycogen.  The  distribution  of 
the  glycogen  was  not  observed  to  differ  from  that  in  the  adult  animal 
except  that  the  liver  of  the  embryo  does  not  assume  its  glycogen-storing 
function  early.  They  further  draw  the  conclusion  that  the  metabolic 
transformations  of  glycogen  in  the  embryo  and  the  adult  are  entirely 
analogous. 

The  lactic  acid  occurring  in  the  muscular  tissue  of  vertebrates  is 
paralactic  or  sarcolactic  acid, 

H     OH 

I       i 
H-C-C-COOH. 

I       I 
H    H 


MUSCULAR   TISSUE. 


257 


The  reaction  of  an  inactive  living  muscle  is  alkaline,  but  upon  the  death 
of  the  muscle,  or  after  the  continued  activity  of  a  living  muscle,  the 
reaction  becomes  acid,  due  to  the  formation  of  lactic  acid.  There  is  a 
difference  of  opinion  regarding  the  origin  of  this  lactic  acid.  Some 
investigators  claim  it  to  arise  from  the  carbohydrates  of  the  muscle, 
while  others  ascribe  to  it  a  protein  origin. 

Among  the  nitrogenous  extratives  of  muscle,  those  which  are  of  the 
most  interest  in  this  connection  are  creatine  and  the  purine  bases,  xanthine 
and  hypoxanthine.  Creatine  is  found  in  varying  amounts  in  the  muscles 
of  "different  species,  the  muscles  of  birds  having  shown  the  largest  amount. 
It-has  also  been  found  in  the  blood,  the  brain,  in  transudates  and  in  the 
thvroid  gland.  Creatine  may  be  crystalHzed  and  forms  colorless  rhombic 
prisms  (Fig.  82,  below)  which  are  soluble  in  warm  water  and  practically 


Fig.  82. — Creatine. 


insoluble  in  alcohol  and  ether.  Upon  boiling  a  solution  of  creatine  with 
dilute  hydrochloric  acid  it  is  dehydrolyzed  and  its  anhydride  creatinine  is 
formed.  The  theory  that  the  creatine  of  ingested  meat  is  transformed 
into  creatinine  and  excreted  in  the  urine  has  been  proven  untenable 
through  the  researches  of  Folin,  KJercker,  and  Wolf  and  Shaffer.  It  is 
now  known  that  under  normal  conditions  the  ingestion  of  creatine  in  no 
way  influences  the  e.xcretion  of  creatinine.  In  the  case  of  Eck  fistula  dogs, 
however,  London  and  Bolyarskii^  found  ingested  creatine  to  increase  the 
output  of  creatinine  in  the  urine.  This  finding  is  of  importance  as  throw- 
ing light  upon  the  role  of  the  liver  in  creatine  and  creatinine  metabolism. 
In  this  connection  it  is  important  to  note  that  there  is  no  normal  excretion 
of  endogenous  (see  p.  291)  creatine,  a  statement  proven  by  the  fact  that 

'  London  and  Bolyarskii:  Zeit.  phys.  chem.,  62,  465,  1909. 
17 


258 


PHYSIOLOGICAL    CHEMISTRY. 


if  no  creatine  be  ingested  none  will  be  excreted.  Folin^  has  shown  that  the 
main  bulk  of  ingested  creatine  is  retained  in  the  body,  unless  the  diet 
contains  a  large  amount  of  protein  material.  Under  certain  pathological 
conditions  the  urine  may  contain  endogenous  creatine  which  is  probably 
derived  from  the  catabohsm  of  muscular  tissue,  as  Benedict,  Mellanby,  and 
Shaffer  have   suggested. 

Amberg  and  Morrill,-  Sedgwick,^  Rose^  and  Folin^  have  shown  that 
creatine  is  a  normal  constitutent  of  the  urine  of  infants  and  children. 
Folin  explains  this  phenomenon  on  the  basis  of  the  relatively  high  protein 
intake,  whereas  Rose  believes  it  is  due  to  a  peculiar  carbohydrate 
metabolism. 


Fig.  83. — Xanthine. 
After  the  drawings  of^Horbaczewski,  as  represented  in  Neubauer  and  Vogel.     {Ogden.) 

Besides  being  a  normal  constituent  of  muscle,  xanthine  has  been 
found  in  the  brain,  spleen,  pancreas,  thymus,  kidneys,  testicles,  liver, 
and  in  the  urine.  It  may  be  obtained  in  crystalline  form  (Fig.  ^7,,  above), 
but  ordinarily  it  is  amorphous.  Xanthine  is  easily  soluble  in  alkalis,  less 
soluble  in  water  and  dilute  acids,  and  entirely  insoluble  in  alcohol  and 
ether. 

Hypoxanthine  occurs  ordinarily  in  those  tissues  and  fluids  which 
contain  xanthine.  It  has  been  found,  unaccompanied  by  xanthine,  in 
bone  marrow  and  in  milk.  Unlike  xanthine  it  may  be  easily  crystallized 
in  the  form  of  small,  colorless  needles.  It  is  readily  soluble  in  alkalis, 
acids,  and  boiling  water,  less  soluble  in  cold  water  and  practically  insoluble 
in  alcohol  and  ether. 

The  predominating  inorganic  salt  of  muscle  is  potassium  phosphate. 

'  Folin:  HammarslenFetschriJt,  p.  15. 

-  Amberg  and  Morrill:  Jour.  Biol,  client.,  3,  311,   1907. 

^Sedgwick:  Jour.  Am.  Med.  Ass'n,  55,  1178,  1910. 

*  Rose:  Jour.  Biol,  chem.,  10,  265,  igii. 

*  Folin:  Ibid,  11,  253,  1912. 


MUSCULAR   TISSUE. 


59 


Besides  this  salt  we  have  present  chlorides  and  sahs  of  sodium,  calcium, 
magnesium,  and  iron.     Sulphates  are  also  present  in  traces. 

Mendel  and  Saiki  have  made  some  interesting  observations  upon  the 
chemical  composition  of  nan-striated  or  smooth  (involuntary)  mammalian 
muscle,  such  as  the  urinary  bladder  and  the  muscular  coat  of  the  stomach 
of  the  pig.  Ilypoxanthine  was  found  to  be  the  y)redominant  purine  base 
present.  Creatine  and  paralactic  acid  were  also  isolated.  These  investi- 
gators were  iinable  to  demonstrate,  definitely,  the  presence  of  glycogen 
in  the  non-striated  muscles  studied,  but  state  that  "the  tissues  possess  the 
property  of  transforming  glycogen  in  the  characteristic  enzymatic  way." 
The  most  important  part  of  their  investigation  consists  in  a  rather  complete 
analysis  of  the  inorganic  constituents  of  these  muscles.  A  notable  differ- 
ence in  the  relative  distribution  of  the  various  inorganic  constituents  was 
observed,  a  difference  which,  according  to  the  authors,  "can  be  accounted 
for  in  part  only  by  an  admixture  of  lymph. ' '  The  comparative  composition 
of  the  inorganic  portion  of  striated  and  non-striated  muscle  and  of  blood 
serum  for  comparison  is  shown  in  the  appended  table: 


Per  loo  parts  of  fresh  muscle. 


K,0 


Na,0 


Fe,0. 


CaO  MgO 


CI 


p,o, 


H,0 


Non-striated     muscle     (Mendel     and 
Saiki) 0.081    0.328    o.oii  0.044  0.007  0.171!  0-184    80.6 


Skeletal  muscle  (Katz) 0.306,  0.210 

Blood  serum  (Abderhalden) 0.0271  0.425 


0.008^0.0110.0470.048    0.487    72.9 
io.oi2  0.004  0.363    0.020    91.8 


An  interesting  comparative  study  of  the  ash  of  the  smooth  muscle  of 
the  stomach  of  the  frog  and  the  striated  muscle  from  the  same  animal  was 
very  recently  reported  by  Meigs  and  Ryan.^  Their  data  indicate  "that 
smooth  muscle  contains  somewhat  less  potassium  and  phosphorus  and 
somewhat  more  sodium  and  chlorine  than  the  striated  muscle  of  the  same 
animal,  but  that  the  differences  in  these  respects  between  the  two  tissues 
are  not  by  any  means  so  marked  as  has  sometimes  been  supposed." 
Their  average  figures  for  each  type  of  muscle  follow: 


Muscle. 


Per  100  parts  of  fresh  muscle. 


Na       Fe    I  Ca 


Mg 


CI    I    S 


Solids   H,0 


Striated 0.350  0.054  o.oio    0.028  0.030  0.155  0.066  0.141    20.13  79.87 

Smooth '0.325  0.073  0.0007  0.004  0.013  0.137  0.1200.1611  17  .70:82  .30 


'  Meigs  and  Ryan:  Journal  of  Biological  Chemistry,  11,  401,  1912. 


26o 


PHYSIOLOGICAL   CHEMISTRY, 


The  preparation  from  which  the  above  data  for  smooth  muscle  were 
obtained  were  shown  by  histological  examination  to  consist  in  large 
part  of  smooth  muscle  fibers. 

Muscular  tissue  is  said  to  contain  a  reddish  pigment  called  myo- 
hcBmatin,  which  is   a   derivative   of  haemoglobin. 

The  so-called  "fatigue  substances"  of  muscle  are  carbon  dioxide, 
paralactic  acid,  and  potassium  dihydrogen  phosphate. 

The  ordinar)^  commercial  "meat  extract"  is  composed  principally 
of  the  water-soluble  constituents  of  muscle  and  contains  practically  nothing 
of  nutritive  value.  The  protein  material  to  which  meat  owes  its  value  as  an 
article  of  diet  is  ordinarily  practically  all  removed  in  the  preparation  of 
the  extract.  Occasionally  some  preparations  are  found  to  contain  pro- 
teose, which  is  formed  from  the  meat  proteins  in  the  process  of  preparation. 

The  structural  formulas  of  some  of  the  nitrogenous  extractives  of 
muscle  are  as  follows: 

NH,  HN CO 


HX=    C 


HN=C 


N.CCHJ.CH^.COOH. 

Creatine,  C4H9N3O2. 

M ethyl- guanidine  acetic  acid. 


N.CCHJ.CH, 

Creatinine,  C4H7N3O. 
Creatine  anhydride. 


NH, 

1 
C=0 

I 
NH, 

Urea^  CON.H4. 


CH,.NH, 


CH2.SO3OH 


Taurine,  C2H7NSO3. 
Amino-ethyl-sulphonic  acid. 


O 


CO 


(CH3)3.N 


CH,— CH  .   OH— CH2 

Carnitine,"  C-HisNOs. 
/■-trimethyloxybutyrobetaine. 

Carnosine,  CgHj^N^Oj. 

Neosine,  CgH^NOj. 

Novaine,  C^Hj^NO,. 

Ignotine,  CgHi.N.Og. 

Phosphocarnic  acid,  C,oHj7N305  or  C10H1-N3O5. 

Inosinic  acid,  (HO)2.PO.O.CH2(CHOH)3.CH:(C5H3N,0). 

The  following  extractives  as  a  group  are  called  purine  bodies.  Their 
formulas,  together  with  that  of  purine  from  which  they  are  derived  and 
the  hypothetical  "purine  nucleus"  follow: 


MUSCULAR  TISSUE. 

N=CH  *N— C" 

I     !  II 

HC     C— NH  ,C     C'— N^ 


261 


— C  —  N 

Purine,  C6H4N4. 

HN— CO 

I       I 
HC     C— NH 


/ 


CH 


.,N-C-N„ 

Purine  Nucleus. 

HN— CO 

I       I 
OC     C— NH 


N— C— N 

HVPOXANTHINE,    C5H4N4O. 

t-oxypurine. 

HN— CO 
OC     C— NH 


CH 


HN— C— N 

Xanthine,  CSH4N4OC 
2-t-dioxy  purine. 

N=C.NH, 

I       I 
HC     C— NH 


>C0 


.CH 


HN— C— NH 

Uric  Acid,  C5H4N4O3. 
2-i>-i-trioxy  purine. 


N— C— N^ 

Adenine,  CsHjNs. 
6-atnitiopurine. 


HN— CO 

I  I 
H^N.C     C— NH 

II  11        \ 


/ 


CH 


N— C— H 

Guanine,  C5H.iN.iO. 
2-amino-6-oxy  purine. 


Experiments  on  Muscular  Tissue. 

I.  Experiments  on  "Living"  Muscle. 

I.  Preparation  of  Muscle  Plasma  (Halliburton). — Wash  out 
the  blood  vessels  of  a  freshly  killed  rabbit  with  0.9  per  cent  sodium 
chloride.  This  can  best  be  done  by  opening  the  abdomen  and  inserting 
a  cannula  into  the  aorta.  Now  remove  the  skin  from  the  lower  limbs, 
cut  away  the  muscles  and  divide  them  into  very  small  pieces  by  means 
of  a  meat  chopper.  Transfer  the  pieces  of  muscle  to  a  mortar  and  grind 
them  with  clean  sand  and  a  little  5  per  cent  magnesium  sulphate.  Filter 
off  the  salted  muscle  plasma  and  make  the  following  tests: 

{a)  Reaction. — Test  the  reaction  to  litmus  phenolphthalein  and  congo 
red.     What  is  the  reaction  of  this  fresh  muscle  plasma  ? 

(b)  Fractional  Coagulation. — Place  a  Httle  muscle  plasma  in  a  test- 
tube  and  arrange  the  apparatus  for  fractional  coagulation  as  explained 


262  PHYSIOLOGICAL    CHEMISTRY. 

on  page  106.  Raise  the  temperature  very  carefully  from  30°  C.  and 
note  any  changes  which  may  occur  and  the  exact  temperature  at  which 
such  changes  take  place.  When  the  first  protein  (para-myosinogen) 
coagulates  filter  it  off  and  then  heat  the  clear  filtrate  as  before,  being 
careful  to  note  the  exact  temperature  at  which  the  next  coagulation 
(myosinogen)  occurs.  There  will  probably  be  a  preliminary  opalescence 
in  each  case  before  the  real  coagulation  occurs.  Therefore  do  not 
mistake  the  real  coagulation-point  and  filter  at  the  wrong  time.  What 
are  the  coagulation  temperatures  of  these  two  proteins  ?  Which  protein 
was  present  in  greater  amount  ? 

(c)  Formation  of  the  Myosin  Clot. — Dilute  a  portion  of  the  plasma 
with  3  or  4  times  its  volume  of  water  and  place  it  on  a  water-bath  or 
in  an  incubator  at  35°  C.  for  several  hours.  A  typical  myosin  clot  should 
form.  Note  the  muscle  serum  surrounding  the  clot.  Now  test  the 
reaction.  Has  the  reaction  changed,  and  if  so  to  what  is  the  change 
due  ?     Make  a  test  for  lactic  acid.     What  do  you  conclude  ? 

2.  Preparation  of  Muscle  Plasma  (v.  Fiirth). — Remove  the  blood- 
free  muscles  of  a  rabbit  as  explained  above.  Finely  divide  by  means  of  a 
meat  chopper  and  grind  in  a  mortar  with  a  little  clean  sand  and  some 
0.9  per  cent  sodium  chloride.  Wrap  portions  of  the  muscle  in  muslin 
and  press  thoroughly  by  means  of  a  tincture  press  or  lemon  squeezer. 
Filter  and  make  the  tests  according  to  the  directions  given  in  the  last 
experiment. 

3.  "Fuchsin-frog"  Experiment. — Inject  a  saturated  aqueous  solu- 
tion of  Fuchsin  "S"  into  the  lymph  spaces  of  a  frog  two  or  three  times 
daily  for  one  or  two  days,  in  this  way  thoroughly  saturating  the  tissues 
with  the  dye.  Pith  the  animal  (insert  a  heavy  wire  or  blunt  needle  through 
the  occipito  atlantoid  membrane),  remove  the  skin  from  both  hind  legs 
and  expose  the  sciatic  nerve  in  one  of  them.  Insert  a  small  wire  hook 
through  the  jaws  of  the  frog  and  suspend  the  animal  from  an  ordinary 
clamp  or  iron  ring.  Pass  electrodes  under  the  exposed  sciatic  nerve, 
and  after  tying  the  other  leg  to  prevent  any  muscular  movement,  stimulate 
the  exposed  nerve  by  means  of  make  and  break  shocks  from  an  induction 
coil.  The  stimulated  leg  responds  by  pronounced  muscular  contractions, 
whereas  the  tied  leg  remains  inactive.  Continue  the  stimulation  until  the 
muscles  are  fatigued.  The  muscular  activity  has  caused  the  production 
of  lactic  acid  and  this  in  turn  has  reacted  with  the  injected  fuchsin  to 
cause  a  pink  or  red  color  to  develop.  The  muscles  of  the  inactive  leg 
still  remain  unchanged  in  color. 

The  normal  color  of  the  Fuchsin  "S"  when  injected  was  red,  but  upon 
being  absorbed  it  became  colorless  through  the  action  of  the  alkalinity 
of  the  blood.     Upon  stimulating  the  muscles,  however,  as  above  explained, 


MUSCULAR    TISSUE.  263 

lactic  acid  was  formed  and  this  acid  reacted  with  the  fuchsin  and  again 
produced  the  original  color  of  the  dye. 

II.  Experiments  on  "Dead"  Muscle. 

1.  Preparation  of  Myosin. — Take  25  grams  of  finely  divided  lean 
beef  which  has  been  carefully  washed  to  remove  blood  and  lymph  constit- 
uents and  place  it  in  a  beaker  with  10  per  cent  sodium  chloride.  Stir 
occasionally  for  several  hours.  Strain  oflf  the  meat  pieces  by  means  of 
cheese  cloth,  filter  the  solution  and  saturate  it  with  sodium  chloride  in 
substance.  Filter  off  the  precipitate  of  myosin  and  make  the  tests  as  given 
below.  This  filtration  will  proceed  very  slowly.  Myosin  collects  as  a 
film  on  the  sides  of  the  filter  paper  and  may  be  removed  and  tested  before 
the  entire  volume  of  fluid  has  been  filtered.  If  this  precipitate  remains 
for  any  length  of  time  on  the  paper  in  contact  with  the  air  it  will  become 
transformed  into  the  protean  myosan.  Test  the  myosin  precipitate  as 
follows : 

(a)  Solubility. — Try  its  solubility  in  the  ordinary  solvents.  Is  myosin 
an  albumin  or  a  globulin  ? 

(b)  Xanthoproteic  Reaction. — See  page  97. 

(c)  Coagulation  Test. — Suspend  a  little  of  the  myosin  in  water  in  a 
test-tube  and  heat  to  boiling  for  a  few  moments.  Now  remove  the  sus- 
pended material  and  try  its  solubility  in  10  per  cent  sodium  chloride. 
What  property  does  this  experiment  show  myosin  to  possess  ? 

Test  the  filtrate  from  the  original  myosin  precipitate  as  follows: 

(a)  Biuret   Test. — What  does  this  show? 

(b)  Place  a  little  of  the  solution  in  a  test-tube  and  heat  to  boiling. 
At  the  boiling-point  add  a  drop  of  dilute  acetic  acid  and  filter.  Test 
this  filtrate  for  proteose  with  picric  acid.  Is  any  proteose  present? 
Saturate  another  portion  of  the  filtrate  with  ammonium  sulphate  and 
test  for  peptone  in  the  usual  way.  (see  page  120)  Do  you  find  any 
peptone?  From  your  experiments  on  "living"  and  ''dead"  muscle  what 
are  your  ideas  regarding  the  proteins  of  muscle  ? 

2.  Preparation  of  Glycogen. — Grind  a  few  oysters  or  scallops^  in  a 
mortar  with  sand.  Transfer  to  an  evaporating  dish,  add  water,  and  boil 
for  20  minutes.  Note  the  opalescence  of  the  solution.  At  the  boiling- 
point  faintly  acidify  with  acetic  acid.  Why  is  this  acid  added  ?  Filter, 
and  divide  the  filtrate  into  two  parts.  Test  one  part  of  the  filtrate  as 
follows : 

(a)  Iodine  Test. — To  50  c.c.  of  the  solution  in  a  test-tube  add  5-10 
drops  of  iodine  solution  and  2-3  drops  of  10  per  cent  sodium  chloride. 

*  Glycogen  may  also  be  prepared  from  the  liver  of  an  animal  which  has  been  fed  a  high 
carbohydrate  diet  for  1-2  days  previously.  The  best  yield  of  glycogen  can,  however,  generally 
be  obtained  from  scallops. 


264  PHYSIOLOGICAL   CHEMISTRY. 

What  do  you  observe  ?     Is  this  similar  to  the  iodine  test  upon  any  other 
body  with  which  we  have  had  to  deal  ? 

If  difl&culty  is  experienced  in  securing  a  satisfactory  iodine  test  proceed 
as  follows:  Make  equal  volumes  of  glycogen  solution  acid  in  reaction 
with  hydrochloric  acid.  Boil  one  solution  to  hydrolyze  the  glycogen. 
Add  equal  volumes  of  iodine  solution  to  each  and  note  the  more  pro- 
nounced iodine  reaction  in  the  unhydrolyzed  solution. 

(b)  Reduction  Test. — Does  the  solution  reduce  Fehling's  solution  ? 

(c)  Hydrolysis  oj  Glycogen. — Add  10  drops  of  concentrated  hydro- 
chloric acid  to  10  c.c.  of  the  solution  and  boil  for  10  minutes.  Cool  the 
solution,  neutralize  with  solid  potassium  hydroxide  and  test  with  Fehling's 
solution.  Does  it  still  fail  to  reduce  Fehling's  solution?  If  you  find  a 
reduction  how  can  you  prove  the  identity  of  the  reducing  substance  ? 

{d)  Influence  oj  Saliva. — ^Place  5  c.c.  of  the  solution  in  a  test-tube, 
add  5  drops  of  saliva  and  place  on  the  water-bath  at  40°  C.  for  10  minutes. 
Does  this  now  reduce  Fehling's  solution  ? 

To  the  second  part  of  the  glycogen  filtrate  add  3-4  volumes  of  95  per 
cent  alcohol.  Allow  the  glycogen  precipitate  to  settle,  decant  the  super- 
natant fluid,  and  filter  the  remainder.  Heat  the  glycogen  on  a  water- 
bath  to  remove  the  alcohol,  then  subject  it  to  the  following  tests: 

{a)  Solubility. — Try  its  solubility  in  the  ordinary  solvents. 

(h)  Iodine  Test. — Place  a  small  amount  of  the  glycogen  in  a  depression 
of  a  test-tablet  and  add  2-3  drops  of  dilute  iodine  solution  and  a  trace  of 
a  sodium  chloride  solution.  The  same  wine-red  color  is  observed  as  in 
the  iodine  test  upon  the  glycogen  solution. 

Separation  of  Extractives  from  Muscle. 

I.  Creatine. — Dissolve  about  10  grams  of  a  commercial  extract 
of  meat  in  200  c.c.  of  warm  water.  Precipitate  the  inorganic  con- 
stituents by  neutral  lead  acetate,  being  careful  not  to  add  an  excess 
of  the  reagent.  Write  the  equations  for  the  reactions  taking  place 
here.  Allow  the  precipitate  to  settle,  then  filter  and  remove  the  excess 
of  lead  in  the  warm  filtrate  by  hydrogen  sulphide.  Filter  while  the 
solution  is  yet  warm,  evaporate  the  clear  filtrate  to  a  syrup,  and  allow  it  to 
stand  at  least  48  hours  in  a  cool  place.  Crystals  of  creatine  should  form 
at  this  point.  Examine  under  the  microscope  (Fig.  82,  page  257).  Treat 
the  syrup  with  200  c.c.  of  88  per  cent  alcohol,  stir  well  with  a  glass  rod  to 
bring  all  soluble  material  into  solution,  and  then  filter.  The  purine  bases 
have  been  dissolved  and  are  in  the  filtrate,  whereas  the  creatine  crystals 
were  insoluble  in  the  88  per  cent  alcohol  and  remain  on  the  filter  paper. 
Wash  the  crystals  with  88  per  cent  alcohol,  then  remove  them  and  bring 


MUSCULAR   TISSUE. 


26q 


them  into  solution  in  a  little  hot  water.  Decolorize  the  solution  by  animal 
charcoal  and  concentrate  it  to  a  small  volume.  Allow  the  solution  to 
cool  and  note  the  separation  of  colorless  crystals  of  creatine.  Examine 
these  crystals  under  the  microscope  and  compare  them  with  those  rejjro- 
duced  in  Fig.  82,  page  257. 

2.  Hypoxanthine. — Evaporate  the  alcoholic  filtrate  from  the  creatine 
to  remove  the  alcohol.  Make  the  solution  ammoniacal  and  add  am- 
moniacal  silver  nitrate  until  precipitation  ceases.  The  precipitate  con- 
sists principally  of  hypoxanthine  silver  and  xanthine  silver.  Collect  these 
silver  salts  on  a  filter  paper  and  wash  them  with  water.  Place  the  pre- 
cipitate and  paper  in  an  evaporating  dish  and  boil  for  one  minute  with 
nitric  acid  having  a  specific  gravity  of  i.i.     Filter  while  hot  through  a 


Fig.  84. — Hypoxanthine  Silver  Nitr.\te. 
(Drawn  from  a  student  preparation  by  Mr.  E.  F.  Hirsch). 


double  paper,  wash  with  the  same  strength  of  nitric  acid  and  allow  the 
solution  to  cool.  By  this  treatment  with  nitric  acid  hypoxanthine  silver 
nitrate  and  xanthine  silver  nitrate  have  been  formed.  The  former  is 
insoluble  in  the  cold  solution  and  separates  on  standing.  After  standing 
several  hours  filter  off  the  hypoxanthine  silver  nitrate  and  wash  \\\\.\\ 
water  until  the  wash-water  is  only  slightly  acid  in  reaction.  Examine 
the  crystals  of  hypoxanthine  silver  nitrate  under  the  microscope  and 
compare  them  with  those  in  Fig.  84,  above.  Now  wash  the  crystals 
from  the  paper  into  a  beaker  with  a  little  water  and  warm  the  liquid. 
Remove  the  silver  by  hydrogen  sulphide  and  filter.  By  this  means 
hypoxanthine  nitrate  has  been  formed  and  is  present  in  the  filtrate.  Con- 
centrate on  a  water-bath  to  drive  off  hydrogen  sulphide  and  render  the 
solution  slightly  alkaline  with  ammonia.     Warm  for  a  time,  to  remove 


266 


PHYSIOLOGICAL    CHEMISTRY. 


the  free  ammonia,  filter,  concentrate  the  filtrate  to  a  small  volume  and 
allow  it  to  stand  in  a  cool  place.  Hypoxanthine  should  crystallize  in 
small  colorless  needles.     Examine  the  crystals  under  the  microscope. 

3.  Xanthine. — To  the  filtrate  from  the  above  experiment  containing 
the  xanthine  silver  ?iitrate  add  ammonia  in  excess.  (The  crystalline  form 
of  xanthine  silver  nitrate  is  shown  in  Fig.  85,  below.)  A  brownish-red 
precipitate  of  xanthine  silver  forms.  Treat  this  suspended  precipitate 
with  hydrogen  sulphide  (do  not  use  an  excess  of  hydrogen  sulphide), 
warm  the  mixture  for  a  few  moments  and  filter  while  hot.     Concentrate 


Fig.  85. — Xanthixe  Silver  Nitrate. 

the  filtrate  to  a  small  volume  and  put  away  in  a  cool  place  for  crystalli- 
zation (Fig.  83,  p.  258).  To  obtain  xanthine  in  crystalline  form  special 
precautions  are  generally  necessary.  Evaporate  the  solution  to  dryness. 
Make  the  following  tests  on  the  crystals  or  residue: 

(a)  Xanthine  Test. — Place  about  one-half  of  the  crystalline  or  amor- 
phous material  in  a  small  evaporating  dish,  add  a  few  drops  of  concen- 
trated nitric  acid  and  evaporate  to  dryness  very  carefully  on  a  water-bath. 
The  yellow  residue  upon  moistening  with  caustic  potash  becomes  red  in 
color  and  upon  further  heating  assumes  a  purplish-red  hue.  Now  add  a 
few  drops  of  water  and  warm.  In  this  way  a  yellow  solution  results 
which  yields  a  red  residue  upon  evaporation.  How  does  this  differ  from 
the  Murexide  test  upon  uric  acid  ? 

{b)  WeideVs  Reaction.— By  gently  heating  bring  the  remainder  of  the 
xanthine  crystals  or  residue  into  solution  in  bromine-water.  Evaporate 
the  solution  to  dryness  on  a  water-bath.  Remove  the  stopper  from  an 
ammonia  bottle  and  by  blowing  across  the  mouth  of  the  bottle  direct  the 
fumes  of  ammonia  so  that  they  come  in  contact  with  the  dry  residue. 
Under  these  conditions  the  presence  of  xanthine  is  shown  by  the  residue 


MUSCULAR   TISSUE.  267 

assuming  a  red  color.  A  somewhat  brighter  color  may  be  obtained  by 
using  a  trace  of  nitric  acid  with  the  bromine-water.  By  the  use  of  this 
modification,  however,  we  may  get  a  positive  reaction  with  bodies  other 
than  xanthine. 

Hurthle's  Experiment. 

Tease  a  very  small  piece  of  frog's  muscle  on  a  microscopical  slide. 
Expose  the  slide  to  ammonia  vapor  for  a  few  moments,  then  adjust  a 
cover  glass,  and  examine  the  muscle  fibers  under  the  microscope. 
Note  the  large  number  of  crystals  of  ammonium  magnesium  phosphate, 

NH,-0 

\ 
Mg-0-P  =  0 

\/ 
O 

distributed  everywhere  throughout  the  muscle  fiber,  thus  demonstrating 
the  abundance  of  phosphates  and  magnesium  in  the  muscle  (Fig.  loi, 
page  319.) 


CHAPTER  XVI. 
NERVOUS  TISSUE. 

In  common  with  the  other  solid  tissues  of  the  body,  nervous  tissue 
contains  a  large  amount  of  water.  The  percentage  of  water  present 
depends  upon  the  particular  form  of  nervous  tissue  but  in  all  forms  it  is 
invariably  greater  in  the  gray  matter  than  in  the  white.  Embryonic 
nervous  tissues  also  contain  a  larger  percentage  of  water  than  the  tissues 
of  adult  life.  The  gray  matter  of  the  brain  of  the  foetus,  for  instance, 
contains  about  92  per  cent  of  water,  whereas  the  gray  matter  of  the  brain 
of  the  adult  contains  but  83-84  per  cent  of  the  fluid. 

Among  the  solid  constituents  of  nervous  tissue  are  proteins,  cho- 
lesterol, cerebrosides  (cerebrin,  etc.),  lecithin,  kephalin,  protagon  {?),  para- 
nucleoprotagon,  nuclein,  neurokeratin,  collagen,  extractives,  and  inorganic 
salts.  The  proteins  are  present  in  the  greatest  amount  and  comprise 
about  50  per  cent  of  the  total  solids.  Three  distinct  proteins,  two  globu- 
lins, and  a  nucleoprotein,  have  been  isolated  from  nervous  tissue.  The 
globulins  coagulate  at  47°  C.  and  70-75°  C,  respectively,  while  the 
nucleoprotein  coagulates  at  56-60°  C.  This  nucleoprotein  contains 
about  0.5  per  cent  of  phosphorus  (Halliburton,  Levene).  Nervous 
tissue  is  composed  of  a  relatively  large  quantity  of  a  variety  of  com- 
pounds which  collectively  may  be  grouped  under  the  term  "lipoid" — 
substances  resembling  the  fats  in  some  of  their  physical  properties 
and  reactions  but  distinct  in  their  composition.  We  will  class  choles- 
terol, the  cerebrosides  and  the  phosphorized  fats  as  lipoids. 

The  consideration  of  lipoids  (or  lipins^)  is  assuming  added  importance. 
These  substances  constitute  one  of  the  two  great  groups  of  tissue  col- 
loids, the  proteins  being  the  remaining  group.  So  far  as  structure  and 
chemical  properties  are  concerned  the  various  classes  of  lipoids  are  entirely 
unlike. 

The  group  of  phosphorized  fats  are  very  important  constituents  'of 
nervous  tissue.  The  best  known  members  of  this  group  are  lecithin, 
protagon  {?)  and  kephalin.  Lecithin  occurs  in  larger  amount  than 
the  other  members  of  the  group,  has  been  more  thoroughly  studied 
than  the  others  and  is  apparently  of  greater  importance.  Upon  decom- 
position lecithin  yields  fatty  acid,   glycero-phosphoric  acid,  and  choline. 

*  Rosenbloom  and  Gies:  Biochemical  Bulletin,  i,  51,  191 1.  The  term  lipoid  was  intro- 
duced by  Overton  (Studien  iiber  die  Narkose,  Jena,  1901,  Gustav  Fischer.) 

268 


NERVOUS   TISSUE.  269 

Each  lecithin  molecule  contains  two  fatty  acid  radicals  which  may  be 
those  of  the  same  or  different  fatty  acids.  Thus  we  have  different 
lecithins  depending  upon  the  particular  fatty  acid  radicals  which  are 
present  in  the  molecule.  The  formula  of  a  typical  lecithin  would  be 
the  following: 

CHO  -C^HgjCO 

I 
CH^O-PO-O-C^H, 

\ 

I  / 

OH  HO 

This  lecithin  would  be  called  distearyl-lecithin  or  choline-distearyl- 
glycero-phosphoric  acid.  Upon  decomposition  the  molecule  splits 
according  to  the  following  reaction: 

C,,H,„NPO«+3H,0->2(C,,H3„0,)  +  C3H„PO«+C,H,,N03. 

Lecithin.  Stearic  acid.       Glycero-phosphoric       Choline. 

acid. 

The  lecithins  are  not  confined  to  the  nervous  tissues  but  are  found  in 
nearly  all  animal  and  vegetable  tissues.  Lecithin  is  a  primary  con- 
stituent of  the  cell.  It  is  soluble  in  chloroform,  ether,  alcohol,  benzene, 
and  carbon  disulphide.  The  chloroform  or  alcohol-ether  solution 
may  be  precipitated  by  acetone.  Lecithin  may  be  caused  to  crystal- 
lize in  the  form  of  small  plates  by  cooling  the  alcoholic  solution  to  a 
low  temperature.  It  has  the  power  of  combining  with  acids  and  bases, 
and  the  hydrochloric  acid  combination  has  the  power  of  forming  a 
double  salt  with  platinic  chloride. 

Choline,  as  was  indicated  above,  is  one  of  the  decomposition  pro- 
ducts of  lecithin.  It  is  trimethyl-oxy ethyl-ammonium  hydroxide  and 
has  the  following  formula : 

CH,.CH,(OH) 

N  =  (CH3)3 

\ 
OH 

Recent  researches  have  shown  that  great  importance  is  to  be  attached 
to  the  detection  of  choHne  in  the  cerebro-spinal  fluid  and  the  blood  in 
certain  cases  of  degenerative  disease  of  the  nervous  system.  In  this 
connection  tests  for  choline  (see  p.  273)  are  of  interest  and  value. 

Protagon,  another  nitrogenous  phosphorized  substance  is  a  body 
over  which  there  has  been  much  discussion.     Upon  decomposition  it 


270  PHYSIOLOGICAL    CHEMISTRY. 

is  said  by  some  investigators  to  yield  cerebrin  and  the  decomposition 
products  of  lecithin.  It  has  been  shown  by  Posner  and  Gies^  as  well 
as  by  Rosenheim  and  Tebb"  that  protagon  is  a  mixture  and  has  no  exist- 
ence as  a  chemical  individual.  Koch^  very  recently  reported  data 
obtained  from  purified  preparations  which  indicate  that  protagon  con- 
tains at  least  three  substances:  "a  phosphatide  containing  cholin,  a 
cerebroside  containing  sugar,  a  complex  combination  of  a  chohn-free 
phosphatide  with  a  cerebroside  to  which  an  ethereal  sulphuric  acid 
group  is  attached."  On  the  basis  of  his  data,  he  beheved  the  term 
protagon  to  have  no  chemical  significance.  He  proposed  the  term  sul- 
phatide.     Koch's  preparation  analyzed  as  follows  (per  cent) : 

Choline  Sugar        Nitrogen        Phosphorus  Sulphur 

i.o  12.0  2.3  1.7  1.9 

He  suggested  the  following  structure: 

O 

11 

Phosphatide  —  O  —  S  —  O  —  Cerebroside 

grouping  jj  grouping 

O 

Kephalin  is  the  third  member  of  the  group  of  phosphorized  fats. 
It  is  precipitated  from  its  acetone-ether  extract  by  alcohol.  It  contains 
about  4  per  cent  of  phosphorus  and  has  been  given  the  formula  C^^B.^^- 
NPOj3.     Kephalin  may  be  a  stage  in  lecithin  metabohsm. 

The  cerebrosides  are  substances  containing  nitrogen  but  no  phos- 
phorus, and  are  important  constituents  of  the  white  matter  of  nerA^ous 
tissue.  Certain  ones  have  also  been  found  in  the  spleen,  pus,  and  in  egg 
yolk.  They  may  be  extracted  from  the  tissue  by  boiling  alcohol  and  are 
insoluble  in  cold  alcohol,  cold  and  hot  ether,  and  in  water  and  dilute 
alkalis.  The  cerebroside  termed  cerebrin  is  a  mixture  containing  phre- 
nosin  (pseudo-cerebrin  or  cerebron),  a  body  yielding  the  carbohydrate 
galactose  on  decomposition. 

Cholesterol,  one  of  the  primary  cell  constituents,  is  present  in  fairly 
large  amount  in  nervous  tissue.  It  is  a  mon-atomic  alcohol  containing 
at  least  one  double  bond  and  possesses  the  formula  C27H^50H  or  C27- 
H^gOH.  There  is  still  some  uncertainty  as  to  the  exact  structure  of 
cholesterol.  It  may  possess  a  terpene  structure.  It  was  formerly  called 
a  "non-saponifiable  fat"  but  since  it  is  not  changed  in  any  way  by 
boiHng  alkalis  it  is  not  a  fat.  It  is  soluble  in  ether,  chloroform,  benzene, 
and  hot  alcohol.     It  crystallizes  in  the  form  of  thin,  colorless,  trans- 

*  Posner  and  Gies:  Journal  0/  Biological  Chemistry,  i,  59,  1905-6. 
^  Rosenheim  and  Tebb:  Journal  of  Physiology,  36  and  37,  1907-8. 
^  Koch:  Journal  0/  Biological  Chemistry,  11,  March,  1912,  Proceedings. 


NERVOUS    TISSUE.  27 1 

parent  plates  (Fig.  43,  p.  166).  Cholesterol  occurs  abundantly  in 
one  form  of  biliary  calculus.  It  has  also  been  found  in  feces,  wool 
fat,  egg  yolk,  and  milk,  frequently  in  the  form  of  its  esters  of  higher 
fatty  acids.  It  is  generally  believed  that  the  cholesterol  present  in  the 
animal  body  has  its  origin  in  the  vegetable  kingdom.  However  evidence 
has  recently  been  submitted'  indicating  a  synthesis  of  cholesterol  under 
certain  conditions  in  the  animal  body. 

Paranucleoprotagon  is  a  phosphorized  substance  originally  isolated 
from  brain  tissue  by  Ulpiani  and  Lelli  and  recently  reinvestigated  by 
Steel   and    Gies.     It   is   said   to   possess   lecithoprotein   characteristics. 

Nervous  tissue  yields  about  i  per  cent  of  ash  which  is  made  up  in 
great  part  of  alkaline  phosphates  and  chlorides. 

Experiments  on  the  Lipoids  of  Nervous  Tissue.' 

1.  Preparation  of  Lecithin. — Treat  the  macerated  brain  of  a 
sheep  with  ether  and  allow  it  to  stand  in  the  cold  for  48-72  hours.  The 
cold  ether  will  extract  lecithin  and  cholesterol.  Filter  and  add  acetone 
to  the  filtrate  to  precipitate  the  lecithin.  Filter  off  the  lecithin  and  test 
it  as  follows: 

(a)  Microscopical  Examination. — Suspend  a  small  portion  in  a 
drop  of  water  on  a  sHde  and  examine  under  the  microscope. 

{h)  Osmic  Acid  Test. — Treat  a  small  portion  with  osmic  acid.  What 
happens  ? 

(c)  Acrolein  Test. — Make  the  acrolein  test  according  to  directions 
on  page  143. 

{d)  "Fusiau"  Test  for  Phosphorus. — Place  some  of  the  lecithin 
prepared  above  in  a  small  porcelain  crucible,  add  a  suitable  amount 
of  a  fusion  mixture  composed  of  potassium  hydroxide  and  potassium 
nitrate  (5  :  i)  and  heat  carefully  until  the  resulting  mixture  is  colorless. 
Cool,  dissolve  the  mass  in  a  little  warm  water,  acidify  with  nitric  acid, 
heat  to  boiling,  and  add  a  few  cubic  centimeters  of  molybdic  solution. 
In  the  presence  of  phosphorus  a  yellow  precipitate  forms.     What  is  it? 

2.  Preparation  of  Cholesterol. — Place  a  small  amount  of  macer- 
ated brain  tissue  under  ether  and  stir  occasionally  for  one  hour.  Filter, 
evaporate  the  filtrate  to  dryness  on  a  water-bath,  and  test  the  cholesterol 
according  to  directions  given  below.     (If  it  is  desired,  the  ether  extract 

*  Klein:  Biochem.  Zeil.,  30,  465,  1910. 

-  Preparation  of  So-called  Protagon. — Macerate  the  brain  of  a  sheep,  treat  with  85  per  cent 
alcohol  and  warm  on  a  water-bath  at  45°  C.  for  two  hours.  Filter  hot  into  a  bottle  or  strong 
flask  and  cool  to  0°  C.  for  one-half  hour  by  means  of  a  freezing  mixture.  By  this  procedure 
both  protagon  and  cholesterol  are  caused  to  precipitate.  Filter  the  cold  solution  rapidly  and 
treat  the  precipitate  on  the  paper  with  ice  cold  ether  to  dissolve  out  the  cholesterol.  The  pro- 
tagon may  now  be  redissolved  in  warm  85  per  cent  alcohol  from  which  solution  it  will  precipi- 
tate upon  cooling. 


272  PHYSIOLOGICAL   CHEMISTRY. 

from  the  so-called  protagon,  or  the  ether-acetone  filtrate  from  the  lecithin 
may  be  used  for  the  isolation  of  cholesterol.  In  these  cases  it  is  simply- 
necessary  to  evaporate  the  solution  to  dryness  on  a  water-bath.)  Upon 
the  cholesterol  prepared  by  either  of  the  above  methods  make  the  fol- 
lowing tests: 

(a)  Microscopical  Examination. — Examine  the  crystals  under  the 
microscope  and  compare  them  with  those  in  Fig.  43,  page  166. 

{b)  Iodine-sulphuric  Acid  Test.- — ^Place  a  few  crystals  of  cholesterol 
in  one  of  the  depressions  of  a  test-tablet  and  treat  with  a  drop  of  concen- 
trated sulphuric  acid  and  a  drop  of  a  very  dilute  solution  of  iodine.  A 
play  of  colors,  consisting  of  violet,  blue,  green,  and  red,  results. 

(c)  The  Liehermann-Burchard  Test. — Dissolve  a  few  crystals  of 
cholesterol  in  2  c.c.  of  chloroform  in  a  dry  test-tube.  Now  add  10 
drops  of  acetic  anhydride  and  1-3  drops  of  concentrated  sulphuric  acid. 
The  solution  becomes  red,  then  blue,  and  finally  bluish-green  in  color. 

{d)  Salkowski's  Test. — Dissolve  a  few  crystals  of  cholesterol  in  a 
little  chloroform  and  add  an  equal  volume  of  concentrated  sulphuric 
acid.  A  play  of  colors  from  bluish-red  to  cherry-red  and  purple  is 
noted  in  the  chloroform,  while  the  acid  assumes  a  marked  green 
fluorescence. 

{e)  Schiff's  Reaction. — To  a  little  cholesterol  in  an  evaporating 
dish  add  a  few  drops  of  Schiff's  reagent.^  Evaporate  to  dryness  over 
a  low  flame  and  observe  the  reddish-violet  residue  which  changes  to  a 
bluish-violet. 

(/)  Phosphorus.- — Test  for  phosphorus  according  to  directions  given 
on  page  271.     Is  phosphorus  present? 

3.  Preparation  of  Cerebrin. — Treat  the  macerated  brain  tissue, 
in  a  flask,  with  95  per  cent  alcohol  and  boil  on  a  water-bath  for  one- 
half  hour,  keeping  the  volume  constant  by  adding  fresh  alcohol  as  needed. 
Filter  the  solution  hot  and  stand  the  cloudy  filtrate  away  for  twenty-four 
hours.  (If  the  filtrate  is  not  cloudy  concentrate  it  upon  the  water-bath 
until  it  is  so.)     Filter  off  the  cerebrin  and  test  it  as  follows: 

{a)  Microscopical  Examination. — Suspend  a  small  portion  in  a 
drop  of  water  on  a  slide  and  examine  under  the  microscope. 

{h)  Solubility. — Try  the  solubility  of  cerebrin  in  the  usual  solvents 
and  in  hot  and  cold  alcohol  and  hot  and  cold  ether. 

(c)  Phosphorus. — Test  for  phosphorus  according  to  directions  on 
page  271.     How  does  the  result  compare  with  that  on  lecithin? 

{d)  Place  a  little  cerebrin  on  platinum  foil  and  warm.  Note  the 
odor. 

'  Schifif's  reagent  consists  of  a  mixture  of  three  volumes  of  concentrated  sulphuric  acid  and 
one  volume  of  10  per  cent  ferric  chloride. 


NERVOUS   TISSUE.  273 

(e)  Hydrolysis  of  Cerehrin. — ^Place  the  remaining  cerebrin  in  a  small 
evaporating  dish,  add  equal  volumes  of  water  and  dilute  hydrochloric 
acid,  and  boil  for  one  hour.  Cool,  neutralize  with  solid  potassium 
hydroxide,  filter,  and  test  with  Fehling's  solution.  Is  there  any  reduction, 
and  if  so  how  do  you  explain  it  ? 

4.  Tests  for  Choline,  {a)  Rosenheim's  Periodide  r«5/.— Prepare 
an  alcoholic  extract  of  the  iluid  under  examination,  and  after  evaporation, 
apply  Rosenheim's  iodo-potassium  iodide  solution^  to  a  little  of  the 
residue.  In  a  short  time  dark  brown  plates  and  prisms  of  choline  periodide 
begin  to  form  and  may  be  detected  by  means  of  the  microscope.  Oc- 
casionally they  are  large  enough  to  be  visible  to  the  naked  eye.  They 
somewhat  resemble  crystals  of  haemin  (see  p.  211).  If  the  slide  be 
permitted  to  stand,  thus  allowing  the  fluid  to  evaporate,  the  crystals 
will  disappear  and  leave  brown  oily  drops.  They  will  reappear,  how- 
ever, upon  the  addition  of  fresh  iodine  solution,  v.  Stanek  claims 
that  this  choline  compound  has  the  formula  CjIIj^NOLIg. 

(b)  Rosenheim's  Bismuth  Test. — Extract  the  fluid  under  examination 
with  absolute  alcohol,  evaporate,  and  re-extract  the  residue.  Repeat 
the  extraction  several  times.  Dissolve  the  final  residue  in  2-3  c.c.  of 
water  and  add  a  drop  of  Kraut's  reagent.^  Choline  is  indicated  by  the 
appearance  of  a  bright  brick-red  precipitate. 

'  Prepared  by  dissolving  2  grams  of  iodine  and  6  grams  of  potassium  iodide  in  100  c.c. 
water. 

^Dissolve  272  grams  of  potassium  iodide  in  water  and  add  80  grams  of  bismuth  sub- 
nitrate  dissolved  in  200  grams  of  nitric  acid  (sp.  gr.  1.18).  Permit  the  potassium  nitrate  to 
crystallize  out,  then  filter  it  off  and  make  the  filtrate  up  to  i  liter  with  water. 


18 


CHAPTER  XVII. 

URINE :  GENERAL  CHARACTERISTICS  OF  NORMAL  AND 
PATHOLOGICAL  URINE. 

Volume. — The  volume  of  urine  excreted  by  normal  individuals 
during  any  definite  period  fluctuates  within  very  wide  limits.  The 
average  output  for  twenty-four  hours  is  placed  by  German  writers 
between  1500  and  2000  c.c.  This  value  is  not  strictly  applicable  to  con- 
ditions in  America,  however,  since  it  has  been  found  that  the  average 
normal  excretion  of  the  adult  male  American  falls  within  the  lower 
values  of  1000-1200  c.c.  The  volume-excretion  is  influenced  greatly 
by  the  diet,  particularly  by  the  ingestion  of  fluids. 

Certain  pathological  conditions  cause  the  output  of  urine  for  any 
definite  period  to  depart  very  decidedly  from  the  normal  output.  Among 
the  pathological  conditions  in  which  the  volume  of  urine  is  increased 
above  normal  are  the  following:  Diabetes  mellitus,  diabetes  insipidus, 
certain  diseases  of  the  nervous  system,  contracted  kidney,  amyloid 
degeneration  of  the  kidney,  and  in  convalescence  from  acute  diseases  in 
general.  Many  drugs  such  as  calomel,  digitalis,  acetates,  and  salicylates 
also  increase  the  volume  of  the  urine  excreted.  A  decrease  from  the 
normal  is  observed  in  the  following  pathological  conditions:  Acute 
nephritis,  diseases  of  the  heart  and  lungs,  fevers,  diarrhoea,  and  vomiting. 

Color. ^Normal  urine  ordinarily  possesses  a  yellow  tint,  the  depth 
of  the  color  being  dependent  in  part  upon  the  density  of  the  fluid.  The 
color  of  normal  urine  is  due  principally  to  a  pigment  called  urochrome: 
traces  of  hcemato porphyrin,  urobilin,  and  uroeryihrin  have  also  been 
detected.  Under  pathological  conditions  the  urine  is  subject  to  pro- 
nounced variations  in  color  and  may  contain  many  varieties  of  pigments. 
Under  such  circumstances  the  urine  may  vary  in  color  from  an  ex- 
tremely light  yellow  to  a  very  dark  brown  or  black.  Vogel  has  constructed 
a  color  chart  which  is  of  some  value  for  purposes  of  comparison.  The 
nature  and  origin  of  the  chief  variations  in  the  urinary  color  are  set 
forth  in  tabular  form  by  Halliburton  as  follows: 

274 


URINE. 


275 


Color. 


Nearly  colorless 


Cause  of  Coloration. 


Pathological  Condition. 


Dilution,     or    diminution     of 
normal  pigments. 


Nervous  conditions:  hy- 
druria,  diabetes  insipidus, 
granular  kidney. 


Dark  vellow  to  l)ro\vn-red.. 


Increase  of  normal,  or  oc- 
currence of  pathological,  pig- 
ments. 


.\cute  febrile  diseases. 


Milkv 


Fat  globules Chyluria. 


Pus  corpuscles. 


Purulent     diseases     of     the 
urinar)-  tract. 


Orange . 


Excreted  drugs Santonin,  chr}sophanic  acid. 


Red  or  reddish. 


Hematopx)rphyrin 

Unchanged  hjemoglobin. 


Haemorrhages,    or    ha?moglo- 
binuria. 


Pigments    in    food    (logwood, 
madder,  bilberries,  fuchsin). 


Brown  to  brown-black. 


Ha;matin 

Small  haemorrhages. 

Methsemoglobin 

Methaemoglobinuria. 

Melanin 

Melanotic  sarcoma. 

Hydrochinon  and  catechol Carbolic-acid  poisoning. 


Greenish-yellow,  greenish-     Bile-pigments, 
brown,  approaching  black. 


Jaundice. 


Dirty  green'  or  blue A  dark-blue  scum  on  surface, 

i  with  a  blue  deposit,  due  to  an 
I  excess  of  indigo-forming  sub- 
I    stances. 


Cholera,  tA'phus;  seen  espe- 
cially when  the  urine  is 
putrefying. 


Brown-yellow  to  red-brown,  I  Substances  contained  in  senna, 
becoming  blood-red  upon  1  rhubarb  and  chelidonium 
adding  alkalis.  I    which  are  introduced  into  the 

I    system. 


Transparency. — Normal  urine  is  ordinarily  perfectly  clear  and 
transparent  when  voided.  On  standing  for  a  variable  time,  however,  a 
cloud  (nubecula)  consisting  principally  of  nucleoprotein  or  mucoid  (see 
p.  308)  and  epithelial  cells  forms.  A  turbidity  due  to  the  precipitation  of 
phosphates  is  normally  noted  in  urine  passed  after  a  hearty  meal. 
The  urine  obtained  2-3  hours  after  a  meal  or  later  is  ordinarily  free  from 
turbidity.  Permanently  turbid  urines  ordinarily  arise  from  pathological 
conditions. 

Odor. — The  odor  of  normal  urine  is  of  a  faint,  aromatic  type.  The 
bodies  to  which  this  odor  is  due  are  not  well  known,  but  it  is  claimed  by  some 
investigators  to  be  due,  at  least  in  part,  to  the  presence  of  minute  amounts 
of  certain  volatile  orsranic  acids.     When  the  urine  undergoes  decom- 


'  This  dirty  green  or  blue  color  also  occurs  after  the  use  of  methylene  blue  in  the  organism. 


276  PHYSIOLOGICAL    CHEMISTRY, 

position,  e.  g.,  in  alkaline  fermentation,  a  very  unpleasant  ammoniacal 
odor  is  evolved.  All  urines  are  subject  to  such  decomposition  if  allowed 
to  stand  for  a  sufficiently  long  time.  Under  normal  conditions  the  urine 
very  often  possesses  a  peculiar  odor  due  to  the  ingestion  of  some  certain 
drug  or  vegetable.  For  instance,  cubebs,  copaiba,  myrtol,  saffron,  tolu, 
and  turpentine  each  imparts  a  somewhat  specific  odor  to  the  urine.  After 
the  ingestion  of  asparagus,  the  urine  also  possesses  a  typical  odor. 

Frequency  of  Urination. — The  frequency  of  urination  varies 
greatly  in  different  individuals  but  in  general  is  dependent  upon  the 
amount  of  fluid  in  the  bladder.  In  pathological  conditions  an  inflam- 
matory affection  of  the  urinary  tract  or  any  disturbance  of  the  innerva- 
tion of  the  bladder  will  influence  the  frequency.  Affections  of  the  spinal 
cord  which  lead  to  an  increased  irritability  of  the  bladder  or  a  weakening 
of  the  sphincter  will  result  in  increasing  the  frequency  of  urination. 

Reaction. — The  mixed  twenty-four  hour  urinary  excretion  of  a 
normal  individual  ordinarily  possesses  an  acid  reaction  to  litmus.  This 
acidity  is  now  believed  to  be  due  to  the  presence  of  various  acidic  radicals 
and  not  to  the  presence  of  sodium  di-hydrogen  phosphate  as  was  formerly 
held  (see  Phosphates,  p.  317).  This  conclusion  is  reinforced  by  the 
observation  that  urine  may  be  divided  into  two  portions,  one  part  con- 
sisting almost  entirely  of  inorganic  matter,  including  practically  all  of 
the  phosphates  and  having  an  alkaline  reaction,  the  other  containing 
practically  all  of  the  organic  substances  and  no  phosphates  and  having 
an  acid  reaction.  The  acidity  imparted  to  the  urine  by  any  particular 
acid  depends  entirely  upon  the  extent  to  which  the  acid  is  dissociable, 
since  it  is  the  hydrogen  ion  which  is  responsible  for  the  acid  reaction. 

The  composition  of  the  food  is  perhaps  the  most  important  factor 
in  determining  the  reaction  of  the  urine.  The  reaction  ordinarily  varies 
considerably  according  to  the  time  of  day  the  urine  is  passed.  For 
instance,  for  a  variable  length  of  time  after  a  meal  the  urine  may  be 
neutral  or  even  alkaline  in  reaction  to  litmus,  owing  to  the  claim  of  the 
gastric  juice  upon  the  acidic  radicals  to  further  the  formation  of  hydro- 
chloric acid  for  use  in  carrying  out  the  digestive  secretory  function.  This 
change  in  reaction  is  known  as  the  alkaline  tide  and  is  common  to  per- 
fectly healthy  individuals.  The  urine  may  also  become  temporarily 
alkaline  in  reaction  to  litmus,  as  the  result  of  ingesting  alkaline  car- 
bonates or  certain  salts  of  tartaric  and  citric  acids  which  may  be  trans- 
formed into  carbonates  within  the  organism.  Normal  urine  upon  stand- 
ing for  some  time  becomes  alkaline  in  reaction  to  litmus,  owing  to  the 
inception  of  alkaline  or  ammoniacal  fermentation  through  the  agency  of 
micro-organisms.  This  fermentation  has  no  especial  diagnostic  value 
except  in  cases  where  the  urine  has  undergone  this  change  within  the 


URINE. 


277 


organism  and  is  voided  in  the  decomposed  slate.  Ammoniacal  fermenta- 
tion is  ordinarily  due  to  cystitis  or  occurs  as  the  result  of  infection  in  the 
process  of  catheterization.  A  microscopical  examination  of  such  urine 
(Fig.  86,  below)  shows  the  presence  of  ammonium  magnesium  phosphate 
crystals,  amorphous  phosphates,  and  not  infrequently  ammonium  urate. 


\Vr ••  v,^»^^  \    i  •'.  ,J  V'"'^,  ■■■>-«  "''.  '-T'  "J'  '  ?.  I 


Fig.  86. — Deposit  in  Ammonla.cal  Fermentation. 
a,  Acid  ammonium  urate;  b,  ammonium  magnesium  phosphate;  c,  bacteria. 

Occasionally  a  urine  which  possesses  a  normal  acidity  when  voided, 
upon  standing  instead  of  undergoing  ammoniacal  fermentation  as  above 
described  will  become  still  more  strongly  acid  in  reaction.  Such  a 
phenomenon  is  termed  acid  jermentation.     Accompanying  this  increased 


—\Si 


Fig.  87. — Deposit  in  Acid  Fermentation. 
a.  Fungus;  b,  amorphous  sodium  urate;  c,  uric  acid;  d,  calcium  oxalate. 

acidity  there  is  ordinarily  a  deepening  of  the  tint  of  the  urinary  color. 
Such  urines  may  contain  acid  urates,  uric  acid,  jungi,  and  calcium  oxalate 
(Fig,  87,  above).  On  standing  for  a  sufficiently  long  time  any  urine 
which  exhibits  acid  fermentation  wall    ultimately  change   in  reaction, 


2/8 


PHYSIOLOGICAL    CHEMISTRY. 


due  to  the  inception  of  alkaline  fermentation,  and  will  show  the  microscop- 
ical deposits  characteristic  of  such  a  urine. 

Specific  Gravity. — The  specific  gravity  of  the  urine  of  normal 
individuals  varies  ordinarily  between  1.015  and  1.025.  This  value  is 
subject  to  wide  fluctuations  under  various  conditions.  For  instance, 
following  copious  water-  or  beer-drinking  the  specific  gravity  may  fall 
to  1.003  or  lower,  whereas  in  cases  of  excessive  perspiration  it  may  rise 
as  high  as  1.040  or  even  higher.  Where  a  very  accurate  determination 
of  the  speciiic  gravity  is  desired  use  is  commonly  made 
of  the  pvknometer  or  of  the  Westphal  hydrostatic  balance. 
These  instruments,  however,  are  not  suited  for  clinical 
use.  The  clinical  method  of  determining  the  specific 
gravity  is  by  means  of  a  urinometer  (Fig.  88).  This 
affords  a  very  rapid  method  and  at  the  same  time  is 
sufficiently  accurate  for  clinical  purposes.  The  urino- 
meter is  always  calibrated  for  use  at  a  specific  tem- 
perature and  the  observations  made  at  any  other  tem- 
perature must  be  subjected  to  a  certain  correction  to 
obtain  the  true  specific  gravity.  In  making  this  cor- 
rection one  unit  oj  the  last  order  is  added  to  the  ob- 
served specific  gravity  for  every  three  degrees  above  the 
normal  temperature  and  subtracted  for  every  three  de- 
grees below  the  normal  temperature.  For  instance,  if 
in  using  a  urinometer  calibrated  for  15°  C.  the  specific 
gravity  of  a  urine  having  a  temperature  of  21°  C.  is 
determined  as  1.018  it  is  necessary  to  add  to  the  observed 
specific  gravity  two  units  of  the  third  order  to  obtain  the 
real  specific  gravity  of  the  urine.  Therefore  the  true 
specific  gravity,  at  15°  C,  of  a  urine  having  a  specific 
gravity  of  1.018  at  21°  C.  is  1.018 -1-0.002  =  1.020. 
Fig.  88.— Urin-  Pathologically,  the  specific  gravity  may  be  subjected 
oMETER  AND  Cyl-  to  Very  wide  variations.  This  is  especially  true  in  dis- 
eases of  the  kidneys.  In  acute  nephritis  ordinarily  the 
urine  is  concentrated  and  of  a  high  specific  gravity,  where.as  in  chronic 
nephritis  the  reverse  conditions  are  more  apt  to  prevail.  In  fact,  under 
most  conditions,  whether  physiological  or  pathological,  the  specific 
gravity  of  the  urine  is  inversely  proportional  to  the  volume  excreted. 
This  is  not  true  of  diabetes  mellitus,  however,  where  the  volume  of 
urine  is  large  and  the  specific  gravity  is  also  high,  owing  to  the  sugar 
contained  in  the  urine. 

The  amount  of  solids  eliminated  in  the  excretion  for  twenty-four 
hours  may  be  roughly  calculated  by  means  oi Long's  coefficient,  i.  e.,  2.6. 


,1 


URINE.  279 

The  solid  content  of  1000  c.c.  of  urintis  obtained  by  mulliplyin«^  the 
last  two  figures  of  the  specific  gravity  observed  at  25°  C.  by  2.6.  To 
determine  the  amount  of  solids  excreted  in  twenty-four  hours  if  the 
volume  was  11 20  c.c.  and  the  specific  gravity  was  1.018  the  calculation 
would  be  as  follows: 

(a)   18X2.6  =  46.8  grams  of  solid  matter  in  1000  c.c.  of  urine. 

46.8X1120 
(0)  -*        -=  !52.4  grams  of  solid  matter  in  11 20  c.c.  of  urine. 

The  coefficient  of  Haser  (2.33)  which  has  been  in  use  for  years 
probably  gives  values  that  are  inaccurate  for  conditions  existing  in 
America.  This  coefficient  was  calculated  on  the  basis  of  the  specific 
gravity  determined  at  a  temperature  of  15°  C. 

Freezing-point  (Cryoscopy).— The  freezing-point  of  a  solution 
depends  upon  the  total  number  of  molecules  of  solid  matter  dissolved 
in  it.  The  determination  of  the  osmotic  pressure  by  this  method  has 
recently  come  to  be  of  some  clinical  importance,  particularly  as  an  aid 
in  the  diagnosis  of  kidney  disorders.  In  this  connection  it  is  best  to 
collect  the  urine  from  each  kidney  separately  and  determine  the  freezing- 
point  in  the  individual  samples  so  collected.  By  this  means  considerable 
aid  in  the  diagnosis  of  renal  diseases  may  be  secured.  The  fluids  most 
frequently  examined  cryoscopically  are  the  blood  (see  p.  194)  and  the 
urine.  The  freezing-point  is  denoted  by  A.  The  value  of  A  for 
normal  urine  varies  ordinarily  between  —1.3°  and  —2.3°  C,  the  freezing- 
point  of  pure  water  being  taken  as  0°.  A  is  subject  to  very  wide  fluctua- 
tions under  unusual  conditions.  For  instance,  following  copious  water- 
or  beer-drinking  A  may  have  as  high  a  value  as  —0.2°  C,  whereas  on  a 
diet  containing  much  salt  and  deficient  in  fluids  the  value  of  A  may  be 
lowered  to  —3°  C.  or  even  lower.  The  freezing-point  of  normal  blood 
is  generally  about  —0.56°  C.  and  is  not  subject  to  the  wide  variations 
noted  in  the  urine,  because  of  the  tendency  of  the  organism  to  maintain 
the  normal  osmotic  pressure  of  the  blood  under  all  conditions.  Variations 
between  —0.51°  and  —0.62°  C.  may  be  due  entirely  to  dietary  conditions, 
but  if  any  marked  variation  is  noted  it  can,  in  most  cases,  be  traced  to  a 
disordered  kidney  function. 

Freezing-point  determinations  may  be  made  by  means  of  the  Beck- 
mann-Heidenhain  apparatus  (Fig.  89)  or  the  Zikel  pektoscope.  The 
Beckmann-Heidenhain  apparatus  consists  of  the  following  parts:  A 
strong  battery  jar  or  beaker  (C)  furnished  with  a  metal  cover  which  is 
provided  with  a  circular  hole  in  its  center.  This  strong  glass  vessel 
serves  to  hold  the  freezing  mixture  by  means  of  which  the  temperature 
of  the  fluid  under  examination  is  lowered.  A  large  glass  tube  (B) 
designed  as  an  air-jacket,  and  formed  after  the  manner  of  a  test-tube  is 


28o 


PHYSIOLOGICAL   CHEMISTRY. 


introduced  through  the  central  aperture  in  the  metal  cover  and  into  this 

air-jacket  is  lowered  a  smaller  tube  (A)  containing  the  fluid  to  be  tested. 

A  very  delicate  thermometer  (D),  graduated  in  hundredths  of  a  degree 

is  introduced  into  the  inner  tube  and  is  held  in 
place  by  means  of  a  cork  so  that  the  mercury 
bulb  is  immersed  in  the  fluid  under  examination 
but  does  not  come  in  contact  with  any  glass 
surface.  A  small  platinum  wire  stirrer  serves 
to  keep  the  fluid  under  examination  well  mixed 
while  a  larger  stirrer  is  used  to  manipulate  the 
freezing  mixture.  (Rock  salt  and  ice  in  the  pro- 
portion 1 : 3  form  a  very  satisfactory  freezing 
mixture.) 

In  making  a  determination  of  the  freezing- 
point  of  a  fluid  by  means  of  the  Beckmann- 
Heidenhain  apparatus  proceed  as  follows:  Place 
the  freezing  mixture  in  the  battery  jar  and  add 
water  (if  necessary)  to  secure  a  temperature  not 
lower  than  3°  C.  Introduce  the  fluid  to  be 
tested  into  tube  A,  place  the  thermometer  and 
platinum  wire  stirrer  in  position,  and  insert  the 
tube  into  the  air-jacket  which  has  previously 
been  inserted  through  the  metal  cover  of  the 
battery  jar.  Manipulate  the  two  stirrers  in 
order  to  insure  an  equalization  of  temperature 
and  observe  the  course  of  the  mercury  column 
of  the  thermometer  very  carefully.  The  mer- 
cury will  gradually  fall  and  this  gradual  lower- 
ing of  the  temperature  will  be  followed  by  a 
sudden  rise.     The  point  at  which  the  mercury 

He^dexi^i'nFre^ezing-point  rests  after  this  sudden  rise  is  the  freezing-point. 

Apparatus.    {Long.)  -phis   rise   is    due  to  the  fact  that  previous  to 

Z),  a  delicate  thermometer;  ^    .,     .         , 

C,  the  containing  jar;  B,  the  freezing,  a  fluid  IS  always  more  or  less  over- 
.rwtrwhXt'Su^e  ^ofe'i  and  the  thermometer  temporarily  regis- 
to  be  observed  is  placed,  ters  a  temperature  somewhat  below  the  freezing- 
Two  stirrers  are  shown,  one        .  a        i       n    •  i    r  i  i 

for  the  cooling  mixture  in  the  pomt.  As  the  fluid  freezes,  howcver,  there  IS  a 
jar  arid  one  for  the  expen-  ^        sudden  change  in  the  temperature  of  the 

mental  mixture.  j  o  i. 

liquid  and  this  change  is  imparted  to  the  ther- 
mometer and  causes  the  rise  as  indicated.  It  occasionally  occurs  that 
the  fluid  under  examination  is  very  much  over-cooled  and  does  not  freeze. 
Under  such  circumstances  a  small  piece  of  ice  is  introduced  into  it  by 
means  of  the  side  tube  noted  in  the  figure.     This  so-called  "inocula- 


URINE.  281 

tion"  causes  the  fluid  to  freeze  instantaneously.  (For  details  of  the 
method  of  determining  the  freezing-point  consult  standard  works  on 
physical  or  organic  chemistry.) 

Electrical  Conductivity. — The  electrical  conductivity  of  the  urine 
is  dependent  upon  the  number  of  inorganic  molecules  or  ions  present, 
and  in  this  diff'ers  from  the  freezing-point  which  is  dependent  upon  the 
total  number  of  molecules  both  inorganic  and  organic  which  are  in 
solution.  The  conductivity  of  the  urine  has  been  investigated  but 
slightly,  and  this  rather  recently,  but  from  the  data  secured  it  seems  that 
the  value  generally  falls  below  k=o.ot,.  The  conductivity  of  blood 
serum  has  been  determined  as  « =0.012.  Up  to  the  present  time  the 
determination  of  the  electrical  conductivity  of  any  of  the  fluids  of  the 
body  has  been  put  to  very  slight  clinical  use.  Experience  may  show 
the  conductivity  value  to  be  a  more  important  aid  to  diagnosis  than  it 
is  now  considered,  particularly  if  it  is  taken  in  connection  with  the  deter- 
mination of  the  freezing-point.  By  a  combination  of  these  two  methods 
the  portion  of  the  osmotic  pressure  due  respectively  to  electrolytes  and 
non-electrolytes  may  be  determined.  For  a  discussion  of  electrical  con- 
ductivity, the  method  by  wh'ch  it  is  determined,  and  the  principles 
involved  consult  standard  works  on  physical  or  electro-chemistry. 

Collection  of  the  Urine  Sample. — If  any  dependable  data  are 
desired  regarding  the  quantitative  composition  of  the  urine  the  examina- 
tion of  the  mixed  excretion  for  twenty-four  hours  is  absolutely  necessary. 
In  collecting  the  urine  the  bladder  may  be  emptied  at  a  given  hour, 
say  8  A.  M.,  the  urine  discarded  and  all  the  urine  from  that  hour  up 
to  and  including  that  passed  the  next  day  at  8  A.  M.,  saved,  thoroughly 
mixed,  and  a  sample  taken  for  analysis.     Powdered  thymol, 

CH, 


\/OH 

CHg  —  CH  —  CHj, 

is  a  very  satisfactory  preservative  since  the  excess  may  be  removed  by 
filtration,  if  desired,  and  any  small  amount  which  may  go  into  solution 
will  have  no  appreciable  influence  upon  the  determination  of  any  of  the 
urinary  constituents.  It  has  no  reducing  power  and  so  may  safely 
be  used  to  preserve  diabetic  urines.  To  insure  the  preservation  of  the 
mixed  urine  of  the  twenty-four  hour  period  it  is  advisable  to  place  a 
small  amount  of  the  thymol  powder  in  the  urine  receptacle  before  the 
first  fraction  of  urine  is  voided.     In  order  to  further  insure  the  preser- 


282  PHYSIOLOGICAL   CHEMISTRY. 

vation  of  the  urine  the  cleaned  and  dried  urine  receptacle  may  be  rinsed 
with  an  alcoholic  solution  of  thymol  and  subsequently  thoroughly  dried 
before  introducing  the  urine. 

Toluol  is  also  used  for  the  preservation  of  urine. 

In  certain  pathological  conditions  it  is  desirable  to  collect  the  urine 
passed  during  the  day  separately  from  that  passed  during  the  night. 
When  this  is  done  the  urine  voided  between  8  A.  M.  and  8  P.  M.  may 
be  taken  as  the  day  sample  and  that  voided  between  8  P.  M.  and  8  A.  M. 
as  the  night  sample. 

The  qualitative  testing  of  urine  voided  at  random,  except  in  a  few 
specific  instances,  is  of  no  particular  value  so  far  as  giving  us  any  accurate 
knowledge  as  to  the  exact  urinary  characteristics  of  the  individual  is 
concerned.  In  the  great  majority  of  cases  the  qualitative  as  well  as  the 
quantitative  tests  should  be  made  upon  the  mixed  excretion  for  a  twenty- 
four  hour  period  as  well  as  upon  a  night  sample  as  above  described. 


CHAPTER  XVTII. 


URINE:  PHYSIOLOGICAL  CONSTITUENTS/ 


I.  Organic  Physiological  Constituents. 


Urea. 
Uric  acid. 

Creatinine. 
Creatine.  ■ 

Ethereal  sulphuric  acids 

Hippuric  acid. 
Oxalic  acid. 


Indoxyl-sulphuric  acid. 
Phenol-  and  />-cresol-sulphuric  acids. 
Pyrocatechin-sulphuric  acid. 
Skatoxyl-sulphuric  acid. 


Xcutral  sulphur  compounds. 


Allantoin. 


Aromatic  oxvacids . 


Cystine. 

Chondroitin-sulphuric  acid. 
Thiocyanates. 
Taurine  derivatives. 
Oxyproteic  acid. 
Alloxyproteic  acid. 
Uroferric  acid. 

Paraoxyphenyl-acetic  acid. 

Paraoxy phenyl-propionic  acid. 

Homogentisic  acid. 

Uroleucic  acid. 

Oxymandelic  acid. 

Kynurenic  acid. 
Amino  acids. 
Benzoic  acid. 
Neucleoprotein. 
Oxaluric  acid. 

'  It  is  impossible  to  make  any  absolute  classification  of  the  physiological  and  pathological 
constituents  of  the  urine.  A  substance  may  be  present  in  the  urine  in  small  amount  physiolog- 
ically and  be  sufficiently  increased  under  certain  conditions  as  to  be  termed  a  pathological 
constituent.  Therefore  it  depends,  in  some  instances,  upon  the  quantity  of  a  constituent  pres- 
ent whether  it  may  be  correctly  termed  a  physiological  or  a  pathological  constituent. 

*  Normal  constituent  of  urine  of  infants  and  children  (see  p.  258). 

283 


284 


PHYSIOLOGICAL   CHEMISTRY. 


f  Pepsin. 

Enzymes \  Gastric  rennin. 

[  Amylase. 

[  Acetic  acid. 

Volatile  fatty  acids ]  Butyric  acid. 

[  Formic  acid. 

Paralactic  acid. 

Phenaceturic  acid. 

^,       ,     .     ,  ,  f  Glycerophosphoric  acid. 

Pnospnorized  compounds <   t,,        ,  .        . , 

^  ^  [  Pnospnocarmc  acid. 

[  Urochrome. 

Pigments I   Uroblin. 

[  Uroerythrin. 

Ptomaines  and  leucomaines. 

Adenine. 

Guanine. 

Xanthine. 

Epiguanine. 

Episarkine. 

Hypoxanthine. 

Paraxanthine. 

Heteroxanthine. 

i-Methylxanthine. 

2.  Inorganic  Physiological  Constituents. 

Ammonia. 

Sulphates. 

Chlorides. 

Phosphates. 

Sodium  and  potassium. 

Calcium  and  magnesium. 

Carbonates. 

Iron. 

Fluorides. 

Nitrates. 

Silicates. 

Hydrogen  peroxide. 


Purine  Bases, 


NH., 


UREA,  C-O. 


NH, 


URINE, 


28: 


Urea  is  the  principal  end-product  of  the  metabolism  of  protein 
substances.  It  has  been  generally  believed  that  about  90  per  cent 
of  the  total  nitrogen  of  the  urine  was  present  as  urea.  Recently, 
however,  Folin  has  shown  that  the  distribution  of  the  nitrogen  of  the 
urine  among  urea  and  the  other  nitrogen-containing  bodies  present 
depends  entirely  upon  the  absolute  amount  of  the  total  nitrogen 
excreted.  He  found  that  a  decrease  in  the  total  nitrogen  excretion 
was  always  accompanied  by  a  decrease  in  the  percentage  of  the  total 
nitrogen  excreted  as  urea,  and  that  after  so  regulating  the  diet  of  a 
normal  person  as  to  cause  the  excretion  of  total  nitrogen  to  be  reduced 
to  3-4  grams  in  24  hours,  only  about  60  per  cent  oj  this  nitrogen  appeared 


Fig.  90. — Urea. 


in  the  urine  as  urea.  His  experiments  also  seem  to  show  urea  to  be  the 
only  one  of  the  nitrogenous  excretions  which  is  relatively  as  well  as 
absolutely  decreased  as  a  result  of  decreasing  the  amount  of  protein 
metabolized.  This  same  investigator  reports  a  hospital  case  in  which 
only  14.7  per  cent  of  the  total  nitrogen  was  present  as  urea  and  about 
40  per  cent  was  present  as  ammonia.  Morner  had  previously  reported 
a  case  in  which  but  4.4  per  cent  of  the  total  nitrogen  of  the  urine  was 
present  as  urea,  and  26.7  per  cent  was  present  as  ammonia. 

Urea  occurs  most  abundantly  in  the  urine  of  man  and  carnivora 
and  in  somewhat  smaller  amount  in  the  urine  of  herbivora;  the  urine 
of  fishes,  amphibians,  and  certain  birds  also  contains  a  small  amount  of 
the  substance.  Urea  is  also  found  in  nearly  all  the  fluids  and  in  many 
of  the  tissues  and  organs  of  mammals.  The  amount  excreted,  under 
normal  conditions,  by  an  adult  man  in  24  hours  is  about  30  grams; 
women  excrete  a  somewhat  smaller  amount.     The  excretion  is  greatest 


286  PHYSIOLOGICAL    CHEMISTRY. 

in  amount  after  a  diet  of  meat,  and  least  in  amount  after  a  diet  con- 
sisting of  non-nitrogenous  foods;  this  is  due  to  the  fact  that  the  last- 
mentioned  diet  has  a  tendency  to  decrease  the  metabolism  of  the  tissue 
proteins  and  thus  cause  the  output  of  urea  under  these  conditions  to 
fall  below  the  output  of  urea  observed  during  starvation.  The  output 
of  urea  is  also  increased  after  copious  water-  or  beer-drinking.  The 
increase  is  probably  due  primarily  to  the  washing  out  of  the  tissues  of 
the  urea  previously  formed,  but  which  had  not  been  removed  in  the 
normal  processes,  and  secondarily  to  a  stimulation  of  protein  catabolism. 

Urea  may  be  formed  in  the  organism  from  amino  acids  such  as  leucine, 
glycocoll,  and  aspartic  acid:  it  may  also  be  formed  from  ammonium 
carbonate  (NHJjCOg  or  ammonium  carbamate,  H^N.O.CO.NHj. 

There  are  differences  of  opinion  regarding  the  transformation  of  the 
substances  just  named  into  urea,  but  there  is  rather  conclusive  evidence 
that  at  least  a  part  of  the  urea  is  formed  in  the  liver;  it  may  be  formed  in 
other  organs  or  tissues  as  well. 

Urea  crystallizes  in  long,  colorless,  four-  or  six-sided,  anhydrous,  rhom- 
bic prisms  (Fig.  90,  p.  285),  which  melt  at  132°  C.  and  are  soluble  in 
water  or  alcohol  and  insoluble  in  ether  or  chloroform.  If  a  crystal 
of  urea  is  heated  in  a  test-tube,  it  melts  and  decomposes  with  the  libera- 
tion of  ammonia.     The  residue  contains  cyanuric  acid, 

COH 

N       N 

II         I 
HO.C       COH 

\^ 

N 


and  biuret, 


NH3 


c=o 

\ 
NH 

y 

c-o 
NH3 


The  biuret  may  be  dissolved  in  water  and  a  reddish-violet  color  obtained 
by  treating  the  aqueous  solution  with  copper  sulphate  and  potassium 
hydroxide  (see  Biuret  Test,  p.  98).  Certain  hypochlorites  or  hypo- 
bromites  in  alkaline  solution  have  the  power  of  decomposing  urea  into 


URINE. 


2S: 


nitrogen,  carbon  dioxide,  and  water.     Sodium  hypobromite  brings  about 
this  decomposition,  as  follows: 

CO(NH,),  +  3NaOBr-*3NaBr  +  N2  +  CO,  +  2H,0. 

This  property  forms  the  basis  for  a  clinical  quantitative  determination 
of  urea  (sec  page  392). 

Urea  has  the  power  of  forming  crystalline  compounds  with  certain 
acids;  urea  nitrate  and  urea  oxalate  are  the  most  important  of  these 
compounds.  Urea  nitrate,  C0(NH,),.HN03,  crystallizes  in  colorless, 
rhombic  or  six-sided  tiles  (Fig.  91,  below),  which  are  easily  soluble  in 
water.  Urea  oxalate,  2.CO(NH2),.H2C204,  crystallizes  in  the  form 
of  rhombic  or  six-sided  prisms  or  plates  (Fig.  93,  p.  289):  the  oxalate 
dififers  from  the  nitrate  in  being  somewhat  less  soluble  in  water. 


Fig.  91. — Urea  Xitrate. 

A  decrease  in  the  excretion  of  urea  is  observed  in  many  diseases  in 
which  the  diet  is  much  reduced  and  in  some  disorders  as  a  result  of 
alterations  in  metabolism,  e.  g.,  myxoedema,  and  in  others  as  a  result 
of  changes  in  excretion,  as  in  severe  and  advanced  kidney  disease.  A 
pathological  increase  is  found  in  a  large  proportion  of  diseases  which 
are  associated  with  a  toxic  state. 

Experiments   ox  Urea. 


I.  Isolation  from  the  Urine. ^ — Place  800  c.c.  of  urine  in  a  pre- 
cipitating jar,  add  250  c.c.   of  baryta  mixture,-  and  stir  thoroughly. 

'  The  method  based  upon  the  precipitation  by  nitric  acid  is  also  satisfactor)'  (see  Hoppe- 
Seyler's  Handbuch  der  Physiol,  und  Pathol.  Chem.  Anal.,  Eighth  edition,  1909,  p.  145.) 

'  Ban-ta  mixture  consists  of  a  mixture  of  one  volume  of  a  saturated  solution  of  Ba(N03)2 
and  two  volumes  of  a  saturated  solution  of  Ba(OH)2. 


PHYSIOLOGICAL   CHEMISTRY. 


Filter  off  the  precipitate  of  phosphates,  sulphates,  urates,  and  hip- 
purates  and  evaporate  the  filtrate  on  a  water-bath  to  a  thick  syrup.  This 
syrup  contains  chlorides,  creatinine,  organic  salts,  pigments,  and  urea. 
Extract  the  syrup  with  warm  95  per  cent  alcohol  and  filter  again.  The 
filtrate  contains  the  urea  contaminated  with  pigment.  Decolorize  the 
filtrate  by  boiling  with  animal  charcoal,  filter  again,  and  stand  the 
filtrate  away  in  a  cold  place  for  crystallization.  Examine  the  crystals 
under  the  microscope  and  compare  them  with  those  shown  in  Fig.  90, 
page  285. 

2.  Solubility. — Test  the  solubility  of  urea,  prepared  by  yourself  or 
furnished  by  the  instructor,  in  the  ordinary  solvents  (see  p.  27)  and  in 
alcohol  and  ether. 

3.  Melting-point. — Determine  the  melting- 
point  of  some  pure  urea  furnished  by  the  instructor. 
Proceed  as  follows:  Into  an  ordinary  melting- 
point  tube,  sealed  at  one  end,  introduce  a  crystal  of 
urea.  Fasten  the  tube  to  the  bulb  of  a  thermo- 
meter as  shown  in  Fig.  92,  and  suspend  the  bulb 
and  its  attached  tube  in  a  small  beaker  contain- 
ing sulphuric  acid.  Gently  raise  the  temperature 
of  the  acid  by  means  of  a  low  flame,  stirring  the 
fluid  continually,  and  note  the  temperature  at 
which  the  urea  begins  to  melt. 

4.  Crystalline  Form. — Dissolve  a  crystal  of 
pure  urea  in  a  few  drops  of  95  per  cent  alcohol 
and  place  1-2  drops  of  the  alcoholic  solution  on  a 
microscopic  slide.  Allow  the  alcohol  to  evaporate 
spontaneously,  examine  the  crystals  under  the  mi- 
croscope, and  compare  them  with  those  reproduced 
in  Fig.  90,  p.  285.  Recrystallize  a  little  urea  from 
water  in  the  same  way  and  compare  the  crystals 
with  those  obtained  from  the  alcoholic  solution. 

5.  Formation  of  Biuret. — Place  a  small 
amount  of  urea  in  a  dry  test-tube  and  heat  care- 
fully in  a  low  flame.  The  urea  melts  at  132°  C. 
and   liberates   ammonia.     Continue  heating  until 

the  fused  mass  begins  to  solidify.  Cool  the  tube,  dissolve  the  residue 
in  dilute  potassium  hydroxide  solution,  and  add  very  dilute  copper  sul- 
phate solution  (see  p.  98).  The  purplish-violet  color  is  due  to  the  pre- 
sence of  biuret  which  has  been  formed  from  the  urea  through  the  appli- 
cation of  heat  as  indicated.     This  is  the  reaction: 


Fig.  92. — Melting- 
point  Tubes  Fastened 
TO  Bulb  of  Thermo- 
meter. 


NH, 


2  C=0 


URINE. 

NH, 

I 

c=o 


289 


Urea. 


NH  +  NH, 


c=o 


NH3 

Biuret. 


6.  Urea    Nitrate, — Prepare    a    concentrated    solution    of    urea    by 
■dissolving  a  little  of  the  substance  in  a  few  drops  of  water.     Place  a 

drop  of  this  solution  on  a  microscopic  slide,  add  a  drop  of  concentrated 
nitric  acid,  and  examine  under  the  microscope.  Compare  the  crystals 
with  those  reproduced  in  Fig.  91,  p.  287. 

7.  Urea  Oxalate.— To  a  drop  of  a  concentrated  solution  of  urea, 
prepared  as  described  in  the  last  experiment  (6),  add  a  drop  of  a  satu- 


FiG.  93. — Urea  Ox.-u-ate. 

rated   solution   of   oxalic   acid.     Examine   under   the   microscope   and 
compare  the  crystals  with  those  shown  in  Fig.  93,  above. 

8.  Decomposition  by  Sodium  Hypobromite. — Into  a  mixture 
of  3  c.c.  of  concentrated  sodium  hydroxide  solution  and  2  c.c.  of  bro- 
mine water  in  a  test-tube  introduce  a  crystal  of  urea  or  a  small  amount  of 
concentrated  solution  of  urea.  Through  the  influence  of  the  sodium  hypo- 
bromite, NaOBr,  the  urea  is  decomposed  and  carbon  dioxide  and  nitrogen 
are  liberated.  The  carbon  dioxide  is  absorbed  by  the  excess  of  sodium 
hydroxide,  while  the  nitrogen  is  evolved  and  causes  the  marked  effer- 
vescence observed.  This  property  forms  the  basis  for  one  of  the  methods 
in  common  use  for  the  quantitative  determination  of  urea.  Write  the 
19 


290  PHYSIOLOGICAL    CHEMISTRY. 

equation  showing  the  decomposition  of  urea  by  sodium  hypobromite. 
g.  Furfurol  Test. — To  a  few  crystals  of  urea  in  a  small  porcelain 
dish  add  1-2  drops  of  a  concentrated  aqueous  solution  of  furfurol  and 
1-2  drops  of  concentrated  hydrochloric  acid.  Note  the  appearance 
of  a  yellow  color  which  gradually  changes  into  a  purple.  Aliantoin 
also  responds  to  this  test  (see  page  305). 

HN-C=0 

I       1 
URIC  ACID,  O  C     C  -  NH 

^CO. 

HN-C-HN 

Uric  acid  is  one  of  the  most  important  of  the  constituents  of  the 
urine.  It  is  generally  stated  that  normally  about  0.7  gram  is  excreted 
in  24  hours  but  that  this  amount  is  subject  to  wide  variations,  particu- 
larly under  certain  dietary  and  pathological  conditions.  Yery  recently 
it  has  been  shown  that  the  average  daily  excretion  of  uric  acid  for  ten 
men  ranging  in  age  from  19  to  29  years  and  fed  a  normal  mixed  diet 
w^as  0.597  g^2,m,  a  value  somewhat  lower  than  the  generally  accepted 
average  of  0.7  gram  for  such  a  period.  Uric  acid  is  a  diureide  and 
consequently  upon  oxidation  pelds  two  molecules  of  urea.  It  acts 
as  a  weak  dibasic  acid  and  forms  two  classes  of  salts,  neutral  and  acid. 
The  neutral  potassium  and  lithium  urates  are  the  most  easily  soluble 
of  the  alkali  salts;  the  ammonium  urate  is  difl&cultly  soluble.  The 
acid-alkali  urates  are  more  insoluble  and  form  the  major  portion  of  the 
sediment  which  separates  upon  cooling  the  concentrated  urine;  the 
alkaline  earth  urates  are  very  insoluble.  Ordinarily  uric  acid  occurs 
in  the  urine  in  the  form  of  urates  and  upon  acidifying  the  liquid  the 
uric  acid  is  liberated  and  deposits  in  crystalline  form.  .  This  property 
forms  the  basis  of  one  of  the  older  methods  for  the  quantitative  deter- 
mination of  uric  acid  (Heintz  Method,  p.  390). 

Uric  acid  is  very  closely  related  to  the  purine  bases  as  may  be  seen 
from  a  comparison  of  its  structural  formula  with  those  of  the  purine 
bases  given  on  page  261.  According  to  the  purine  nomenclature  it  is 
designated  2-6-8-trioxypurine.  Uric  acid  forms  the  principal  end- 
product  of  the  nitrogenous  metabolism  of  birds  and  scaly  amphibians; 
in  the  human  organism  it  occupies  the  fourth  position  inasmuch  as  here 
urea,  ammonia,  and  creatinine  are  the  chief  end-products  of  nitrogenous 
metabolism.  It  is  generally  said  that  the  relarion  existing  between 
uric  acid  and  urea  in  human  urine  under  normal  conditions  varies  on 
the  average  from  1:40  to  1:100  and  is  subject  to  wider  variations  under 
pathological  conditions;  and  I'urther  th:.t  because  of  the  high  content  of 


PT.ATE  V. 


Uric  Acid  Crystals.     Normal  Color.     (From  Purdy,  after  Peyer.) 


URINE.  291 

uric  acid  in  the  urine  of  new-born  infants  the  ratio  may  be  reduced  to 
1 :  10  or  even  lower.  We  now  know  that  this  ratio  of  uric  acid  to  urea 
is  of  little  significance  under  any  conditions. 

In  man,  uric  acid  probably  results  principally  from  the  destruction 
of  nuclein  material.  It  may  arise  from  nuclein  or  other  purine  material 
ingested  as  food  or  from  the  disintegrating  cellular  matter  of  the  organ- 
ism. The  uric  acid  resulting  from  the  first  process  is  said  to  be  of  ex- 
ogenous origin,  whereas  the  product  of  the  second  form  of  activity  is 
said  to  be  of  endogenous  origin.  As  a  result  of  experimentation,  Siv6n, 
and  Burian  and  Schur,  and  Rockwood  claim  that  the  amount  of  endoge- 
nous uric  acid  formed  in  any  given  period  is  fairly  constant  for  each 
individual  under  normal  conditions,  and  that  it  is  entirely  independent 
of  the  total  amount  of  nitrogen  eliminated.  Recently  FoHn  has  taken 
exception  to  the  statements  of  these  investigators  and  claims  that,  fol- 
lowing a  pronounced  decrease  in  the  amount  of  protein  metabolized, 
the  absolute  quantity  of  uric  acid  is  decreased  but  that  this  decrease  is 
relatively  smaller  than  the  decrease  in  the  total  nitrogen  excretion  and 
that  the  per  cent  of  the  uric  acid  nitrogen,  in  terms  of  the  total  nitrogen, 
is  therefore  decidedly  increased. 

In  birds  and  scaly  amphibians  the  formation  of  uric  acid  is  analo- 
gous to  the  formation  of  urea  in  man.  In  these  organisms  it  is  derived 
principally  from  the  protein  material  of  the  tissues  and  the  food  and  is 
formed  through  a  process  of  synthesis  which  occurs  for  the  most  part 
in  the  liver;  a  comparatively  small  fraction  of  the  total  uric  acid  excre- 
tion of  birds  and  scaly  amphibians  may  result  from  nuclein  material. 
When  pure,  uric  acid  may  be  obtained  as  a  white,  odorless,  and 
tasteless  powder,  which  is  composed  principally  of  small,  transparent, 
crystalHne,  rhombic  plates.  Uric  acid  as  it  separates  from  the  urine 
is  invariably  pigmented,  and  crystallizes  in  a  large  variety  of  character- 
istic forms,  e.  g..  dumb-bells,  wedges,  rhombic  prisms,  irregular  rect- 
angular or  hexagonal  plates,  whetstones,  prismatic  rosettes,  etc.  Uric 
acid  is  insoluble  in  alcohol  and  ether,  soluble  with  diflficulty  in  boiling 
water  (1:1800)  and  practically  insoluble  in  cold  water  (1:39,480,  at 
18°  C).  It  is  soluble  in  alkalis,  alkali  carbonates,  boiling  glycerol, 
concentrated  sulphuric  acid,  and  in  certain  organic  bases  such  as  ethyl- 
amine  and  piperidine.  It  is  claimed  that  the  uric  acid  is  held  in  solu- 
tion in  the  urine  by  the  urea  and  disodium  hydrogen  phosphate  present. 
Uric  acid  possesses  the  power  of  reducing  cupric  hydroxide  in  alkaline 
solution  and  may  thus  lead  to  an  erroneous  conclusion  in  testing  for 
sugar  in  the  urine  by  means  of  Fehling's  or  Trommer's  tests.  A  white 
precipitate  of  cuprous  urate  is  formed  if  only  a  small  amount  of  cupric 
hydroxide  is  present,  but  if  enough  of  the  copper  salt  is  present  the 


292  PHYSIOLOGICAL   CHEMISTRY. 

characteristic  red  or  brownish-red  precipitate  of  cuprous  oxide  is  ob- 
tained. Uric  acid  does  not  possess  the  power  of  reducing  bismuth  in 
alkaline  solution  and  therefore  does  not  interfere  in  testing  for  sugar  in 
the  urine  by  means  of  Boettger's  or  Nylander's  tests. 

In  addition  to  being  an  important  urinary  constituent  uric  acid  is 
normally  present  in  the  brain,  heart,  liver,  lungs,  pancreas,  and  spleen; 
it  also  occurs  in  the  blood  of  birds  and  has  been  detected  in  traces  in 
human  blood  under  normal  conditions. 

Pathologically,  the  excretion  of  uric  acid  is  subject  to  wide  variations, 
but  the  experimental  findings  are  rather  contradictory.  It  may  be 
stated  with  certainty,  however,  that  in  leukaemia  the  uric  acid  output 
is  increased  absolutely  as  well  as  relatively  to  the  urea  output;  under 
these  conditions  the  ratio  between  the  uric  acid  and  urea  may  be  as 
low  as  1:9,  whereas  the  normal  ratio,  as  we  have  seen,  is  i :  50  or  higher. 
In  the  study  of  the  influence  of  X-ray  on  metabolism  Edsall  reached 
some  interesting  conclusions.  He  found  that  the  excretion  of  uric  acid 
is  usually  increased  and  that  in  some  conditions,  particularly  in  leukaemia, 
it  may  be  greatly  increased.  The  excretion  of  total  nitrogen,  phos- 
phates, and  other  substances  may  also  be  considerably  increased. 

Experiments  on  Uric  Acid. 

1.  Isolation  from  the  Urine. — Place  about  200  c.c.  of  filtered 
urine  in  a  beaker,  render  it  acid  with  2-10  c.c.  of  concentrated  hydro- 
chloric acid,  stir  thoroughly,  and  stand  the  vessel  in  a  cold  place  for  24 
hours.  Examine  the  pigmented  crystals  of  uric  acid  under  the  micro- 
scope and  compare  them  with  those  shown  in  Fig.  106,  p.  365  and  PI. 
v.,  opposite  p.  291. 

2.  Solubility. — Try  the  solubility  of  pure  uric  acid,  furnished  by 
the  instructor,  in  the  ordinary  solvents  (see  p.  27)  and  in  alcohol,  ether, 
concentrated  sulphuric  acid  and  in  boiling  glycerol. 

3.  Crystalline  Form  of  Pure  Uric  Acid. — Place  about  100  c.c.  of 
water  in  a  small  beaker,  render  it  distinctly  alkaline  with  potassium 
hydroxide  solution  and  add  a  small  amount  of  pure  uric  acid,  stirring 
continuously.  Cool  the  solution,  render  it  distinctly  acid  with  hydro- 
chloric acid  and  allow  it  to  stand  in  a  cool  place  for  crystallization. 
Examine  the  crystals  under  the  microscope  and  compare  them  with 
those  reproduced  in  Fig.  94,  p.  293. 

4.  Murexide  Test. — To  a  small  amount  of  pure  uric  acid  in. a  small 
evaporating  dish  add  2-3  drops  of  concentrated  nitric  acid.  Evapo- 
rate to  dryness  carefully  on  a  water-bath  or  over  a  very  low  flame.  A 
red  or  yellow  residue  remains  which  turns  purplish-red  after  cooling  the 


URINE. 


293 


dish  and  adding  a  drop  of  very  dilute  ammonium  hydroxide.  The  color 
is  due  to  the  formation  of  murexide.  If  potassium  hydroxide  is  used 
instead  of  ammonium  hydroxide  a  purplish-violet  color  due  to  the  pro- 
duction of  the  potassium  salt  is  obtained.  The  color  disappears  upon 
warming;  with  certain  related  bodies  (purine  bases)  the  color  persists 
under  these  conditions, 

5.  Moreigne's  Reaction. — To  equal  volumes  of  Moreigne's  reagent^ 
and  the  solution  to  be  tested  add  a  few  drops  of  concentrated  potas- 
sium hydroxide.     A  blue  color  indicates  the  presence  of  uric  acid. 

6.  Schiff's  Reaction. — Dissolve  a  small  amount  of  pure  uric  acid 
in  sodium  carbonate  solution  and  transfer  a  drop  of  the  resulting  mix- 


Fro.  94. — Pt.-RE  Uric  .\cid. 

ture  to  a  strip  of  filter  paper  saturated  with  silver  nitrate  solution. 
A  yellowish-brown  or  black  coloration  due  to  the  formation  of  reduced 
silver  is  produced. 

7.  Ganassini's  Test.- — Dissolve  a  small  amount  of  uric  acid  in 
sodium  carbonate.  Precipitate  the  dissolved  uric  acid  by  means  of 
zinc  chloride,  filter  oflf  the  precipitate,  and  permit  it  to  stand  in  contact 
with  the  air.  A  sky-blue  color  will  develop,  a  color  change  which  may 
be  hastened  by  sunlight.  A  similar  reaction  may  be  obtained  by  treat- 
ing the  original  precipitate  with  K2S2O8. 

8.  Influence  upon  Fehling's  Solution. — Dilute  i  c.c.  of  Fehling's 
solution  \\ith  4  c.c.  of  water  and  heat  to  boiling.  Now  add  slowly,  a 
few  drops  at  a  time,  1-2  c.c.  of  a  concentrated  solution  of  uric  acid  in 

*  Moreigne's  reagent  is  made  by  combining  20  grams  of  sodium  tungstate,  10  grams 
of  phosphoric  acid  (sp.  gr.  1.13)  and  100  c.c.  of  water.  Boil  this  mixture  for  twenty  minutes, 
add  water  to  make  the  volume  of  the  solution  equivalent  to  the  original  volume,  and  acidify 
with  hydrochloric  acid. 

*  Ganassini:  Boll,  soc,  1908,  No.  i. 


294  PHYSIOLOGICAL    CHEMISTRY. 

potassium  hydroxide,  heating  after  each  addition.  From  this  experiment 
what  do  you  conclude  regarding  the  possibiHty  of  arriving  at  an  erroneous 
decision  when  testing  for  sugar  in  the  urine  by  means  of  Fehling's  test  ? 
9.  Reduction  of  Nylander's  Reagent. — To  5  c.c.  of  a  solution  of 
uric  acid  in  potassium  hydroxide  add  about  one-half  a  cubic  centimeter 
of  Nylander's  reagent  and  heat  to  boiling  for  a  few  moments.  Do  you 
obtain  the  typical  black  end-reaction  signifying  the  reduction  of  the 
bismuth  ? 

NH      -     CO 

I 

CREATININE,  C  =  NH 

N.(CH3).CH, 

Creatinine  is  the  anhydride  of  creatine  and  is  a  constituent  of  normal 
human  urine.  The  theory  that  creatinine  is  derived  from  the  creatine 
of  ingested  muscular  tissue  as  well  as  from  the  creatine  of  the  muscular 
tissue  of  the  organism  has  recently  been  proven  to  be  incorrect  by  Folin, 
Klercker,  and  Wolf  and  Shaffer.  Shaffer  believes  that  creatinine  is 
the  result  of  some  special  process  of  normal  metabolism  which  takes 
place  to  a  large  extent,  if  not  entirely,  in  the  muscles  and  further  that 
the  amount  of  such  creatinine  elimination,  expressed  in  milligrams  per 
kilogram  body  weight,  is  an  index  of  this  special  process.^  He  further 
states  that  the  muscular  efficiency  of  the  individual  depends  upon  the 
intensity  of  this  process.  Under  normal  conditions  about  i  gram  of 
creatinine  is  excreted  by  an  adult  man  in  24  hours, ^  the  exact  amount 
depending  in  great  part  upon  the  nature  of  the  food  and  decreasing 
markedly  in  starvation.  Very  little  that  is  important  is  known  regarding 
the  excretion  of  creatinine  under  pathological  conditions.  The  creatinine 
content  of  the  urine  is  said  to  be  increased  in  typhoid  fever,  typhus, 
tetanus,  and  pneumonia,  and  to  be  decreased  in  anaemia,  chlorosis, 
paralysis,  muscular  atrophy,  advanced  degeneration  of  the  kidneys,  and 
in  leukaemia  (myelogenous,  lymphatic  and  pseudo).  An  increase  of 
creatinine  was  also  noted  in  diabetes,  an  increase  probably  due  to  the 
creatinine  content  of  the  meat  eaten.  The  greater  part  of  the  data, 
however,  relating  to  the  variation  of  the  creatinine  excretion  under 
pathological  conditions  are  not  of  much  value  since  in  nearly  every 
instance  the  diet  was  not  sufficiently  controlled  to  permit  the  collection 
of  reliable  data.  And  further,  until  the  advent  of  the  Folin  method 
(see  p.  415),  there  was  no  accurate  method  for  the  quantitative  determina- 

'  He  propsoes  to  designate  as  the  "creatinine  coefficient"  the  excretion  of  creatinine-nitro- 
gen  (mgs.)  per  kilogram  of  body  weight. 

^  According  to  Shaffer  the  amount  excreted  by  strictly  normal  individuals  is  between  7 
and  II  milligrams  of  crealinine-nitrogen  per  kilogram  of  body  weight. 


URINK. 


295 


tion  of  creatinine.  ShalTer  has  very  recently  called  attention  to  the  fact 
that  a  low  excretion  of  creatinine  is  found  in  the  urine  of  a  remarkably 
large  number  of  pathological  subjects,  representing  a  variety  of  conditions, 
and  that  it  is  therefore  evident  that  the  excretion  of  an  abnormally  small, 
amount  of  this  substance  is  by  no  means  peculiar  to  any  one  disease. 

Creatinine  crystallizes  in  colorless,  glistening  monoclinic  prisms 
(Fig.  95,  belo\v)  which  are  soluble  in  about  12  parts  of  cold  water;  they  are 
more  solube  in  warm  water  and  in  warm  alcohol.  It  forms  salts  only 
with  strong  mineral  acids.  One  of  the  most  important  and  interesting 
of  the  compounds  of  creatinine  is  creatinine-zinc  chloride,  (C^H7N30)2- 
ZnCU.  which  is  formed  from  an  alcoholic  solution  of  creatinine  upon 


Fig.  95. — Creatinine. 


treatment  with  zinc  chloride  in  acid  solution.  Creatinine  has  the  power 
of  reducing  cupric  hydroxide  in  alkaline  solution  and  in  this  way  may 
interfere  with  the  determination  of  sugar  in  the  urine.  In  the  reduction 
by  creatinine  the  blue  liquid  is  first  changed  to  a  yellow  and  the  formation 
of  a  brownish-red  precipitate  of  cuprous  oxide  is  brought  about  only 
after  continuous  boiling  with  an  excess  of  the  copper  salt.  Creatinine 
does  not  reduce  alkaline  bismuth  solutions  and  therefore  does  not  interfere 
with  Nylander's  and  Boettger's  tests. 

It  has  recently  been  shown  by  Folin  that  the  absolute  quantity  of 
creatinine  eliminated  in  the  urine  on  a  meat-free  diet  is  a  contsant  quantity 
different  for  different  individuals,  but  wholly  independent  of  quantita- 
tive changes  in  the  total  amount  of  nitrogen  eliminated.  Shaffer  has 
very  recently  confirmed  these  findings  and  has  shown  that  the  output  of 
creatinine  under  these  conditions  is  constant  from  hour  to  hour  as  well 
as  from  dav  to  dav. 


296 


PHYSIOLOGICAL    CHEMISTRY. 


According  to  Pekelharing^  muscular  tonus  increases  the  creatinine 
excretion  of  the  individual  whereas  muscular  exertion  does  not. 

Experiments  on  Creatinine. 

I.  Separation  from  the  Urine. — Place  250  c.c.  of  urine  in  a  casserole 
or  beaker,  render  it  alkaline  with  milk  of  lime  and  then  add  CaClj  solution 
until  the  phosphates  are  completely  precipitated.  Filter  off  the  precipi- 
tate, render  the  filtrate  slightly  acid  with  acetic  acid,  and  evaporate  it  to  a 
syrup.  While  still  warm  this  syrup  is  treated  with  about  50  c.c.  of  95-97 
per  cent  alcohol  and  the  mixture  allowed  to  stand  8-12  hours  in  a  cool 
place.  The  precipitate  is  now  filtered  off  and  the  filtrate  treated  with  a 
little  sodium  acetate  and  about  one-half  c.c.  of  acid-free  zinc  chloride 
solution  having  a  specific  gravity  of  1.2.    This  mixture  is  stirred  thoroughly 


Fig. 


Creatinine-zinc  Chloride.     (Salkowski.) 


and  allowed  to  stand  in  a  cold  place  for  48-72  hours.  Creatinine-zinc 
chloride  (Fig.  96,  above)  will  crystalhze  out  under  these  conditions. 
Collect  the  crystals  on  a  filter  paper  and  wash  them  with  alcohol  to 
remove  chlorides.  Now  treat  the  zinc  chloride  compound  with  a  little 
warm  water,  boil  with  lead  oxide  and  filter.  The  filtrate  may  now  be 
decolorized  by  animal  charcoal,  evaporated  to  dryness,  and  the  residue 
extracted  with  strong  alcohol.  (Creatine  remains  undissolved  under 
these  conditions.)  The  alcoholic  extract  of  creatinine  is  now  evapo- 
rated to  incipient  crystallization  and  left  in  a  cool  place  until  crystal- 
lization is  complete.  If  desired  the  crystals  may  be  purified  by  re- 
crystallization  from  water. 

2.  Weyl's  Test. — Take  5  c.c.  of  urine  in  a  test-tube,  add  a  few 
drops  of  sodium  nitro-prusside  and  render  the  solution  alkaline  with 

*  Pekelharing:  Onderzoekingengedaan  in  het  Physiol,  Lab.  te  Utrecht,  Vol.  5,  No.  12,  1911. 


URINE.  297 

potassium  hydroxide  solution.     A  ruby-red   color  results  which  soon 
turns  yellow.     See  Legal's  test  for  acetone,  page  349. 

3.  Salkowski's  Test. — To  the  yellow  solution  obtained  in  Weyl's 
test  above  add  an  excess  of  acetic  acid  and  apply  heat.  A  green  color 
results  and  is  in  turn  displaced  by  a  blue  color.  A  precipitate  of  Prussian 
blue  may  form. 

4.  Jaffe's  Reaction.— Place  5  c.c.  of  urine  in  a  test-tube,  add  an 
aqueous  solution  of  picric  acid  and  render  the  mixture  alkaline  with 
potassium  hydroxide  solution.  A  red  color  is  produced  which  turns 
yellow  if  the  solution  be  acidified.  Dextrose  gives  a  similar  red  color 
but  only  upon  the  application  of  heat.  This  color  reaction  observed 
when  creatinine  in  alkaline  solution  is  treated  with  picric  acid  is  the 
basic  principle  of  FoHn's  colorimetric  method  for  the  quantitative 
determination  of  creatinine  (see  page  415.) 

ETHEREAL  SULPHURIC  ACIDS. 

The  most  important  of  the  ethereal  sulphuric  acids  found  in  the 
urine  are  phenol-sulphuric  acid,  p-cre sol-sulphuric  acid,  indoxyl-sulphuric 
acid,  and  skatoxyl-sidphuric  acid.  Pyrocatechin-sulphuric  acid  also 
occurs  in  traces  in  human  urine.  The  total  output  of  ethereal  sul- 
phuric acid  varies  from  0.09  to  0.62  gram  for  24  hours.  In  health  the 
ratio  of  ethereal  sulphuric  acid  to  inorganic  sulphuric  acid  is  about 
1:10.  These  ethereal  sulphuric  acids  originate  in  part  from  the  phenol, 
cresol,  indole  and  skatole  formed  in  the  putrefaction  of  protein  material 
in  the  intestine.  The  phenol  passes  to  the  liver  where  it  is  conjugated 
to  form  phenol  potassium  sulphate  and  appears  in  this  form  in  the  urine 
whereas  the  indole  and  skatole  undergo  a  preHminary  oxidation  to  form 
indoxyl  and  skatoxyl  respectively  before  their  elimination. 

It  has  generally  been  considered  that  each  of  the  ethereal  sulphuric 
acids  was  formed  principally  in  the  putrefaction  of  protein  material  in 
the  intestine  and  that  therefore  a  determination  of  the  total  ethereal 
sulphuric  acid  content  of  the  urine  was  an  index  of  the  extent  to  which 
these  putrefactive  processes  were  proceeding  within  the  organism. 
Recently,  however,  Folin  has  conducted  a  series  of  experiments  which 
seem  to  show  that  the  ethereal  sulphuric  acid  content  of  the  urine  does 
not  afford  an  index  of  the  extent  of  intestinal  putrefaction,  since  these 
bodies  arise  only  in  part  from  putrefactive  processes.  He  claims  that  the 
ethereal  sulphuric  acid  excretion  represents  a  form  of  sulphur  metabohsm 
which  is  more  in  evidence  upon  a  diet  containing  a  very  small  amount  of 
protein  or  upon  a  diet  containing  absolutely  no  protein.  The  ethereal 
sulphuric  acid  content  of  the  urine  diminishes  as  the  total  sulphur  content 
diminishes  but  the  percentage  decrease  is  much  less.     Therefore  when 


298  PHYSIOLOGICAL    CHEMISTRY. 

considered  from  the  standpoint  of  the  total  sulphuric  acid  content  the 
ethereal  sulphuric  acid  content  is  not  diminished  but  is  increased,  although 
the  total  sulphuric  acid  content  is  diminished.  Folin's  experiments  also 
seem  to  show  that  the  indoxyl  sulphuric  acid  (indoxyl  potassium  sulphate 
or  indican)  content  of  the  urine  does  not  originate  to  any  degree  from  the 
metabolism  of  protein  material  but  that  it  arises  in  great  part  from 
intestinal  putrefaction  and  that  the  excretion  of  indoxyl  sulphuric  acid 
may  alone  be  taken  as  a  rough  index  of  the  extent  of  putrefactive  proc- 
esses within  the  intestine,     Indoxyl  sulphuric  acid, 

CH 

-       /\ 
HC      C-C(0.S03H), 

HC      C      CH 


CH  NH 

therefore,  which  occurs  in  the  urine  as  indoxyl  potassium  sulphate  or 
indican, 

CH 


HC      C  -  C(0.S03K), 

I      II     !! 

HC      C      CH 


CH  NH 

is  clinically  the  most  important  of  the  ethereal  sulphuric  acids. 

Tests  for  Indican.^ 

I.  Jaffe's  Test. — Nearly  fill  a  test-tube  with  a  mixture  composed 
of  equal  volumes  of  concentrated  HCl  and  the  urine  under  exami- 
nation. Add  2-3  c.c.  of  chloroform  and  a  few  drops  of  a  calcium 
hypochlorite  solution,  place  the  thumb  over  the  end  of  the  test-tube 
and  shake  the  tube  and  contents  thoroughly.  The  chloroform  is  colored 
more  or  less,  according  to  the  amount  of  indican  present.  Ordinarily  a 
blue  color  due  to  the  formation  of  indigo-blue  is  produced;  less  frequently 
a  red  color  due  to  indigo-red  may  be  noted. 

Repeat  this  test  on  some  of  this  same  urine  to  which  formaldehyde  has 
been  added.  Is  there  any  variation  in  the  reaction  from  what  you 
previously  obtained  ? 

'  Tlie  urine  should  always  be  examined /re^/f  if  this  is  possible.  In  any  event  formaldehyde 
should  never  be  used  as  a  preservative  for  such  urines  as  are  to  be  examined  for  indican  by 
means  of  any  test  involving  hypochlorite  or  potassium  permanganate.  The  formaldehyde 
through  its  redu(  ing  power  lowers  the  oxidizing  efficiency  of  the  mixture  The  formation  of 
formic  acid  from  the  aldehyde  may  also  interfere. 


URINK.  299 

This  is  the  reaction  (see  also  page  1O9): 
CI  I 


HC      C  -  con 

2      I        II        II         +2O- 
HC      C       CH 


CH  NH 

Indoxyl,  C»H7NO. 

CH  CH 


HC      C  -  CO     O.C-C      CH 

I         II        I  I        II       I       +2H,0 

HC      C      C==C      C      CH 


CH  NH  NH  CH 

Indigo-blur,  Ci;-,HioNuOj. 

2.  Obermayer's  Test. — Nearly  fill  a  test-tube  with  a  mixture  com- 
posed of  equal  volumes  of  Obermayer's  reagent^  and  the  urine  under 
examination.  Add  2-3  c.c.  of  chloroform,  place  the  thumb  over  the  end 
of  the  test-tube  and  shake  thoroughly.  How  does  this  compare  with 
Jaffa's  test  ? 

3.  Giirber's  Reaction. — To  dne  volume  of  the  urine  under  ex- 
amination and  two  volumes  of  concentrated  hydrochloric  acid  in  a 
test-tube  add  2-3  drops  of  a  i  per  cent  solution  of  osmic  acid  and  2-3 
c.c.  of  chloroform  and  shake  the  tube  and  contents  thoroughly.  Com- 
pare the  color  with  those  obtained  in  Jaffe's  and  Obermayer's  tests. 

An  excess  of  osmic  acid  does  not  affect  the  reaction.  Occasionally 
better  results  are  obtained  if  the  solution  of  osmic  acid  is  added  directly 
to  the  urine  before  the  addition  of  the  hydrochloric  acid.  If  the  urine 
under  examination  be  strongly  colored  or  of  high  specific  gravity  it 
should  first  be  treated  with  basic  lead  acetate  (one-eighth  volume). 
The  precipitate  is  then  removed  by  filtration  and  the  resulting  filtrate 
used  in  making  the  test  for  indican. 

4.  Rossi's  Reaction. — To  equal  volumes  of  concentrated  hydro- 
chloric acid  and  the  urine  under  examination,  in  a  test-tube,  add  i 
drop  of  a  10  per  cent  solution  of  ammonium  persulphate  and  2-3  c.c. 
of  chloroform.  Agitate  the  mixture  vigorously  and  note  the  color  of 
the  chloroform.  Compare  this  result  with  those  obtained  in  the  other 
indican  tests. 

5.  Lavelle's  Reaction. — To  10  c.c.  of  urine  in  a  test-tube  add 
2-3  c.c.  of  Obermayer's  reagent^  and  a  similar  volume  of  concentrated 

*  Obermayer's  reagent  is  prepared  by  adding  2-4  grams  of  ferric  chloride  to  a  liter  of  con- 
centrated HCl  (sp.  gr.  1. 19). 


300 


PHYSIOLOGICAL    CHEMISTRY. 


sulphuric  acid.  (During  the  addition  of  the  acid  the  tube  should  be 
held  under  running  water  in  order  that  the  temperature  of  the  mixture 
may  not  rise  too  high.)  Add  2-3  c.c.  of  chloroform,  shake  the  tube 
^dgorously,  and  observe  the  depth  of  color  assumed  by  the  chloroform. 

The  sponsor  for  this  reaction  claims  it  to  be  the  most  satisfactory 
of  the  indican  tests. 

6.  Barberio's  Reaction/ — To  5  c.c.  of  the  urine  in  a  test-tube 
add  2-3  drops  of  a  sodium  nitrite  solution  (i :  2000)  and  mix  well  by  shak- 
ing. Now  add  5  c.c.  of  concentrated  hydrochloric  acid  and  2-3  c.c. 
of  chloroform  and  again  shake.  Note  the  color  of  the  chloroform. 
Compare  this  test  with  tests  i  and  2  on  the  same  urine. 


CO.NH.CH^.COOH. 


flIPPURIC  ACID, 


This  acid  occurs  normally  in  the  urine  of  both  the  carnivora  and 
herbivora  but  is  more  abundant  in  the  urine  of  the  latter.     It  is  formed 


Fig.  97. — HipPURic  Acid. 

by  a  synthesis  of  benzoic  acid  and  glycocoU  which  takes  place  in  the 
kidneys.  The  average  excretion  of  an  adult  man  for  24  hours  under 
normal  conditions  is  about  0.7  gram.  Hippuric  acid  crystallizes  in 
needles  or  rhombic  prisms  (see  Fig.  97,  above)  the  particular  form 
depending  upon  the  rapidity  of  crystallization.  Pure  hippuric  acid 
melts  at  187°  C.  The  most  satisfactory  method  for  the  isolation  of 
hippuric  acid  from  the  urine  in  crystalline  form  is  that  proposed  by 
Roaf   (see  below j.     It  is  easily  soluble  in  alcohol  or  hot  water,  and 

'  Barberio:  Policlinico,  No.  17,  191 1. 


URINE.  301 

only  slightly  soluble  in  ether.  The  output  of  hippuric  acid  is  increased 
in  diabetes  owing  probably  to  the  ingestion  of  much  protein  and  fruit. 
It  is  decreased  in  fevers  and  in  certain  kidney  disorders  where  the  synthetic 
activity  of  the  renal  cells  is  diminished.  Hippuric  acid  may  be  deter- 
mined quantitatively  by  means  of  Dakin's  methods  (see  p.  406). 

Experiments  on  Hippuric  Acid. 

I.  Separation  from  the  Urine,  (a)  First  Method. — Render  500- 
1000  c.c.  of  urine  of  the  horse  or  cow^  alkaline  with  milk  of  lime,  boil 
for  a  few  moments  and  filter  while  hot.  Concentrate  the  filtrate,  over 
a  burner,  to  a  small  volume.  Cool  the  solution,  acidify  it  strongly 
with  concentrated  hydrochloric  acid  and  stand  it  in  a  cool  place  for 
24  hours.  Filter  off  the  crystals  of  hippuric  acid  which  have  formed 
and  wash  them  with  a  little  cold  water.  Remove  the  crystals  from 
the  paper,  dissolve  them  in  a  very  small  amount  of  hot  water  and  per- 
colate the  hot  solution  through  thoroughly  washed  animal  charcoal, 
being  careful  to  wash  out  the  last  portion  of  the  hippuric  acid  solution 
with  hot  water.  Filter,  concentrate  the  filtrate  to  a  small  volume  and 
stand  it  aside  for  crystallization.  Examine  the  crystals  under  the  micro- 
scope and  compare  them  with  those  in  Fig.  97,  page  300.  This  method 
is  not  as  satisfactory  as  Roaf's  method  (see  below). 

(b)  Roafs  Method. — Place  500  c.c.  of  urine  of  the  horse  or  cow^ 
in  a  casserole  or  precipitating  jar  and  add  an  equal  volume  of  a  satu- 
rated solution  of  ammonium  sulphate^  and  7.5  c.c.  of  concentrated 
sulphuric  acid.  Permit  the  mixture  to  stand  for  twenty-four  hours  and 
remove  the  crystals  of  hippuric  acid  by  filtration.  Purify  the  crystals 
by  recrystallization  according  to  the  directions  given  above  under  First 
Method.  Examine  the  crystals  under  the  microscope  and  compare 
them  with  those  given  in  Fig.  97,  p.  300. 

If  sufficient  urine  is  not  available  to  peimit  the  use  of  500  c.c.  a 
smaller  volume  may  be  used  inasmuch  as  it  is  possible,  by  the  above 
technic,  to  isolate  hippuric  acid  in  crystalline  form  from  as  small  a 
volume  as  25-50  c.c.  of  herbivorous  urine.  The  greater  the  amount  of 
ammonium  sulphate  added  the  more  rapid  the  crystallization  until 
at  the  saturation  point  the  crystals  of  hippuric  acid  sometimes  form  in 
about  ten  minutes. 

*  If  urine  of  the  horse  or  cow  is  not  available  human  urine  may  serve  the  purpose  fully 
as  well  provided  means  are  taken  to  increase  its  content  of  hippuric  acid.  This  may  be  con- 
veniently accomplished  by  ingesting  2  grams  of  ammonium  benzoate  at  night.  The  fraction 
of  urine  passed  in  the  morning  will  be  found  to  have  a  high  content  of  hippuric  acid.  The 
ammonium  benzoate  is  in  no  way  harmful.  In  case  ammonium  benzoate  is  not  available 
sodium  benzoate  may  be  substituted. 

*  125  grams  of  solid  ammonium  sulphate  may  be  substituted. 


302  PHYSIOLOGICAL    CHEMISTRY. 

2.  Melting-point. — Determine  the  melting-point  of  the  hippuric 
acid  prepared  in  the  above  experiment  (see  p.  146). 

3.  Solubility. — Test  the  solubility  of  hippuric  acid  in  the  ordinary 
solvents  (page  27)  and  in  alcohol,  and  ether. 

4.  Dehn's  Reaction. — Introduce  about  5  c.c.  of  the  urine  or  the 
solution  under  examination  into  a  test-tube  and  add  sufficient  hypo- 
bromite  solution^  to  impart  to  the  mixture  a  permanent  yellow  color. 
In  the  case  of  urine  enough  hypobromite  should  be  added  to  decom- 
pose the  urea.  Heat  the  mixture  to  boiling  and  note  the  formation 
of  an  orange  or  brown-red  precipitate  if  hippuric  acid  is  present.  If 
the  solution  under  examination  contains  only  a  trace  of  hippuric  acid 
the  solution  will  appear  smoky  and  faintly  red  in  color,  whereas  if  a 
larger  amount  of  the  acid  be  present  the  solution  will  become  opaque 
and  of  an  orange  or  brown-red  color.  In  either  case  after  standing 
for  some  time  the  solution  should  clear  up  and  a  light,  finely  divided 
precipitate  should  be  deposited.  This  precipitate  consists  of  earthy 
phosphates  mixed  with  an  amorphous  orange  or  brown-red  substance 
of  unknown  composition.  (For  some  unknown  reason  this  reaction 
does  not  always  yield  satisfactory  results  even  on  pure  hippuric  acid 
solution.) 

5.  Formation  of  Nitro-Benzene. — To  a  little  hippuric  acid  in  a 
small  porcelain  dish  add  1-2  c.c.  of  concentrated  HNO3  and  evapo- 
rate to  dryness  on  a  water-bath.  Transfer  the  residue  to  a  dry  test- 
tube,  apply  heat,  and  note  the  odor  of  the  artificial  oil  of  bitter  almonds 
(nitro-benzene). 

6.  Sublimation. — Place  a  few  crystals  of  hippuric  acid  in  a  dry 
test-tube  and  apply  heat.  The  crystals  are  reduced  to  an  oily  fluid 
which  solidifies  in  a  crystalline  mass  upon  cooling.  When  stronger 
heat  is  applied  the  liquid  assumes  a  red  color  and  finally  yields  a  sub- 
limate of  benzoic  acid  and  the  odor  of  hydrocyanic  acid. 

7.  Formation  of  Ferric  Salt. — Render  a  small  amount  of  a  solu- 
tion of  hippuric  acid  neutral  with  dilute  potassium  hydroxide.  Now 
add  1-3  drops  of  neutral  ferric  chloride  solution  and  note  the  formation 
of  the  ferric  salt  of  hippuric  acid  as  a  cream  colored  precipitate. 

COOH 
OXALIC  ACID,  I 

COOH 

Oxalic  acid  is  a  constituent  of  normal  urine,  about  0.02  gram  being 
eliminated  in  24  hours.  It  is  present  in  the  urine  as  calcium  oxalate, 
which  is  kept  in  solution  through  the  medium  of  the  acid  phosphates. 
The  origin  of  the  oxalic  acid  content  of  the  urine  is  not  well  under- 

'  See  note  on  p.  392. 


URINE.  303 

Stood.  It  is  eliminated,  at  least  in  part,  unchanged  when  ingested,  there- 
fore since  many  of  the  common  articles  of  diet,  e.  g.,  asparagus,  apples, 
cabbage,  grapes,  lettuce,  spinach,  tomatoes,  etc.,  contain  oxalic  acid  it 
seems  probable  that  thq  ingested  food  supplies  a  portion  of  the  oxalic 
acid  found  in  the  urine.  There  is  also  experimental  evidence  that  part 
of  the  oxalic  acid  of  the  urine  is  formed  within  the  organism  in  the 
course  of  protein  and  fat  metabolism.  It  has  also  been  suggested  that 
oxalic  acid  may  arise  from  an  incomplete  combustion  of  carbohydrates, 
especially  under  certain  abnormal  conditions.  Pathologically,  oxalic 
acid  is  found  to  be  increased  in  amount  in  diabetes  mellitus,  in  organic 
diseases  of  the  liver,  and  in  various  other  conditions  which  are  accom- 
panied by  a  derangement  of  the  oxidation  mechanism.  An  abnormal 
increase  of  oxalic  acid  is  termed  oxaluria.  A  considerable  increase  in 
the  content  of  oxalic  acid  may  be  noted  unaccompanied  by  any  other 
apparent  symptom.  Calcium  oxalate  crystallizes  in  at  least  two  distinct 
forms,  dumb-bells  and  oclahedra  (Fig.  104,  page  363). 

Experiments. 

Preparation  of  Calcium  Oxalate.  First  Method. — Place  200-250 
c.c.  of  urine  in  a  beaker,  add  5  c.c.  of  a  saturated  solution  of  calcium 
chloride,  make  the  urine  slightly  acid  with  acetic  acid,  and  stand  the 
beaker  aside  in  a  cool  place  for  24  hours.  Examine  the  sediment  under 
the  microscope  and  compare  the  crystalline  forms  with  those  shown  in 
Fig.  104,  p.  363. 

Second  Method. — ^Proceed  as  above,  replacing  the  acetic  acid  by  an 
excess  of  ammonium  hydroxide  and  filtering  off  the  precipitate  of  phos- 
phates. 

NEUTRAL  SULPHUR  COMPOUNDS. 

Under  this  head  may  be  classed  such  bodies  as  cystine  (see  p.  80), 
chondroitin-sulphuric  acid,  oxyproteic  acid,  alloxyproteic  acid,  uroferric 
acid,  thiocyanates  and  taurine  derivatives.  The  sulphur  content  of  the 
bodies  just  enumerated  is  generally  termed  loosely  combined  or  neutral 
sulphur  in  order  that  it  may  not  be  confused  with  the  acid  sulphur  which 
occurs  in  the  inorganic  sulphuric  acid  and  ethereal  sulphuric  acid  forms 
Ordinarily  the  neutral  sulphur  content  of  normal  human  urine  is  14-20 
per  cent  of  the  total  sulphur  content. 

NH.CH.NH 

I  I    . 

ALLANTOIN,  OC  CO. 

!  I 

NH.CO  NH 


304 


PHYSIOLOGICAL   CHEMISTRY. 


Allantoin  has  been  found  in  the  urine  of  suckHng  calves  as  well 
as  in  that  of  the  dog  and  cat,  rabbit,  monkey,  horse  and  nian.^  It  has 
also  been  detected  in  the  urine  of  infants  within  the  first  eight  days  after 
birth,  as  well  as  in  the  urine  of  adults.  It  is  mgre  abundant  in  the  urine 
of  women  during  pregnancy.  Underbill  also  reports  the  presence  of 
allantoin  in  the  urine  of  fasting  dogs,  an  observation  which  makes  it 
probable  that  allantoin  is  a  constant  constituent  of  the  urine  of  such 
animals.  Allantoin  is  formed  by  the  oxidation  of  uric  acid  and  the  output 
is  increased  by  the  feeding  of  thymus  or  pancreas  to  lower  animals. 
In  fact  allantoin  is  considered  to  be  the  principal  end-product  of  purine 


Fig.  98. — Allantoin,  from  Cat's  Urine. 

a  and  b,  Forms  in  which  it  crystallized  from  the  urine;  c,  recrystallized  allantoin.     (Drawn 

from  micro-photographs  furnished  by  Prof.  Lafayette  B.  Mendel  of  Yale  University.) 


metabolism  in  such  animals.  Nothwithstanding  certain  evidence^  favor- 
ing this  view  it  is  not  generally  believed  that  allantoin  is  an  important 
end-product  of  purine  metabolism  in  man.  When  pure  it  crystaUizes 
in  prisms  (Fig.  98,  above)  and  when  impure  in  granules  and  knobs. 
Pathologically,  it  has  been  found  increased  in  diabetes  insipidus  and  in 
hysteria  with  convulsions  (Pouchet).  Mendel  and  Dakin*  have  recently 
shown  that  allantoin  is  optically  inactive  notwithstanding  the  fact  that  it 
contains  an  asymmetric  carbon  atom.  This  phenomenon  they  believe  to 
be  due  to  tautomeric  change.  Wiechowski  has  suggested  an  excellent 
method  for  the  quantitative  determination  of  allantoin.* 

*  Wiechowski:  Biochemische  Zeitschtift,  19,  368,  1909. 

^Ascher:  Biochemische  Zeitschrift,  26,  370,  1910;  Fairhall  and  Hawk:  Jour.  Am.  Chem, 
Soc,  34,  546,  1912. 

^Mendel  and  Dakin:  Jour.  Biol.  Chem.,  7,  153,  1910. 

*  Wiechowski:  Biochemische  Zeitschrift,  19,  368,  1909. 


URINE.  305 

Experiments. 

1.  Separation  from  the  Urine/  Meissner's  Method. — Precipi- 
tate the  urine  with  Ijaryta  water.  Neutralize  the  filtrate  carefully 
with  dilute  sulphuric  acid,  filter  immediately,  and  evaporate  the  fil- 
trate to  incipient  crystallization.  Completely  precipitate  this  warm 
fluid  with  95  per  cent  alcohol  (reserve  the  precipitate).  Decant  or 
filter  and  precipitate  the  solution  by  ether.  Combine  the  ether  and 
alcohol  precipitates  and  extract  with  cold  water  or  hot  alcohol;  allantoin 
remains  undissolved.  Bring  the  allantoin  into  solution  in  hot  water  and 
re  crystallize. 

Allantoin  may  be  determined  quantitively  by  the  Paduschka- 
Underhill-Kleiner  method  (see  p.  432)  or  by  Loewi's  method.^ 

2.  Preparation  from  Uric  Acid.— Dissolve  4  grams  of  uric  acid 
in  100  c.c.  of  water  rendered  alkaline  with  potassium  hydroxide.  Cool 
and  carefully  add  3  grams  of  potassium  permanganate.  Filter,  immedi- 
ately acidulate  the  filtrate  with  acetic  acid  and  allow  it  to  stand  in  a  cool 
place  over  night.  Filter  oiT  the  crystals  and  wash  them  with  water. 
Save  the  wash  water  and  filtrate,  unite  them  and  after  concentrating  to 
a  small  volume  stand  away  for  crystalHzation.  Now  combine  all  the 
crystals  and  recrystallize  them  from  hot  water.  Use  these  crystals  in 
the  experiments  which  follow. 

3.  Microscopical  Examination. — Examine  the  crystals  made  in 
the  last  experiment  and  compare  them  with  those  shown  in  Fig.  98, 
page  304. 

4.  Solubility. — Test  the  solubility  of  allantoin  in  the  ordinary 
solvents  (page  27.) 

5.  Reaction. — Dissolve  a  crystal  in  water  and  test  the  reaction  to 
litmus. 

6.  Furfurol  Test. — Place  a  few  crystals  of  allantoin  on  a  test-tablet 
or  in  a  porcelain  dish  and  add  1-2  drops  of  a  concentrated  aqueous 
solution  of  furfurol  and  1-2  drops  of  concentrated  hydrochloric  acid. 
Observe  the  formation  of  a  yellow  color  which  turns  to  a  light  purple  if 
allowed  to  stand.     This  test  is  given  by  urea  but  not  by  uric  acid. 

7.  Murexide  Test. — Try  this  test  according  to  the  directions  given 
on  page  292.     Note  that  allantoin  fails  to  respond. 

8.  Reduction  of  Fehling's  Solution. — Make  this  test  in  the  us  al 
way  (see  p.  32)  except  that  the  boiling  must  be  prolonged  and  excessive. 
Ultimately  the  allantoin  will  reduce  the  solution.  Compare  with  the 
result  on  uric  acid,  page  293. 

'  The  urine  of  the  dog  after  thymus,  pancreas,  or  uric  acid  feeding  may  be  employed. 
*  Archiv  fur  Experimentelle  Pathologie  und  Pharmakologie,  44,  20,  1900. 


306  PHYSIOLOGICAL   CHEMISTRY. 

AROMATIC  OXYACIDS. 

Two  of  the  most  important  of  the  oxyacids  are  paraoxyphenyl-acetic 
acid. 


CH,.COOH, 


OH 

and  paraoxyphenyl-propionic  acid, 


CH,.CH2.C00H. 


OH 

They  are  products  of  the  putrefaction  of  protein  material  and  tyrosine 
is  an  intermediate  stage  in  their  formation.  Both  these  acids  for  the 
most  part  pass  unchanged  into  the  urine  where  they  occur  normally  in 
very  small  amount.  The  content  may  be  increased  in  the  same  manner 
as  the  phenol  content,  in  particular  by  acute  phosphorous  poisoning.  A 
fraction  of  the  total  aromatic  oxyacid  content  of  the  urine  is  in  combi- 
nation with  sulphuric  acid,  but  the  greater  part  is  present  in  the  form 
of  salts  of  sodium  and  potassium. 

Homogentisic  Acid  or  di-oxyphenyl-acetic  acid, 

OH 

CH^.COOH, 


OH 

is  another  important  oxyacid  sometimes  present  in  the  urine.  Under  the 
,  name  glycosuric  acid  it  was  first  isolated  from  the  urine  by  Prof.  John 
Marshall  of  the  University  of  Pennsylvania;  subsequently  Baumann 
isolated  it  and  determined  its  chemical  constitution.  It  occurs  in  cases 
of  alcaptonuria.  A  urine  containing  this  oxyacid  turns  greenish-brown 
from  the  surface  downward  when  treated  with  a  little  sodium  hydroxide 
or  ammonia.  If  the  solution  be  stirred  the  color  very  soon  becomes  dark 
brown  or  even  black.  Homogentisic  acid  reduces  alkaline  copper  solu- 
tions but  not  alkaline  bismuth  solutions.  Uroleucic  acid  is  similar  in 
its  reactions  to  homogentisic  acid. 

Oxymandelic  Acid  or  paraoxyphcnyl-glycolic  acid, 


URINE.  307 

OH 


CH(OH).COOH, 

has  been  detected  in  t|je  urine  in  cases  of  yellow  atrophy  of  the  liver. 
Kynurenic  Acid  or  ^-oxy-;9-quinoline  carbonic  acid, 

CH  COH 

HC      C      C.COOH, 

I        II        I 
HC      C      CH 

\/\/ 
CH  N 

is  present  in  the  urine  of  the  dog  and  has  recently  been  detected  by 
Swain  in  the  urine  of  the  coyote.  To  isolate  it  from  the  urine  proceed 
as  follows:  Acidify  the  urine  with  hydrochloric  acid  in  the  propor- 
tion 1:25.  From  this  acid  fluid  both  the  uric  acid  and  the  kynurenic 
acid  separate  in  the  course  of  24-48  hours.  Filter  oflf  the  combined 
crystalline  deposit  of  the  two  acids,  dissolve  the  kynurenic  acid  in  dilute 
ammonia  (uric  acid  is  insoluble),  and  reprecipitate  it  with  hydrochloric 
acid. 

Kynurenic   acid    may   be    quantitatively    determined   by    Capaldi's 
method.^ 

COOH. 

/ 
BENZOIC  ACID, 


Benzoic  acid  has  been  detected  in  the  urine  of  the  rabbit  and  dog.  It  is 
also  said  to  occur  in  human  urine  accompanying  renal  disorders.  The 
benzoic  acid  probably  originates  from  a  fermentative  decomposition  of  the 
hippuric  acid  of  the  urine. 

Experiments. 

1.  Solubility.— Test  the  solubility  of  benzoic  acid  in  water,  alcohol, 
and  ether. 

2.  Crystalline  Form. — Recrystallize  some  benzoic  acid  from  hot 
water,  examine  the  crystals  under  the  microscope,  and  compare  them 
with  those  reproduced  in  Fig.  99,  p.  308. 

3.  Sublimation. — Place  a  little  benzoic  acid  in  a  test-tube  and  heat 

'  Zeitschri/t  fiir  physiologische  Chemie,  23,  92,  1897. 


?o8 


PHYSIOLOGICAL   CHEMISTRY. 


over  a  flame.     Note  the  odor  which  is  evolved  and  observe  that  the  acid 
sublimes  in  the  form  of  needles. 

4.  Dissolve  a  little  sodium  benzoate  in  water  and  add  a  solution 
of  neutral  ferric  chloride.  Note  the  production  of  a  brownish-yellow 
precipitate  (salicylic  acid  gives  a  reddish-violet  color  under  the  same 
conditions).  Add  ammonium  hydroxide  to  some  of  the  precipitate. 
It  dissolves  and  ferric  hydroxide  is  formed.     Add  a  little  hydrochloric 


Fig.  99. — Benzoic   Acid. 

acid  to  another  portion  of  the  original  precipitate  and  stand  the  vessel 
away  over  night.     What  do  you  observe  ? 

NUCLEOPROTEIN. 

The  nubecula  of  normal  urine  has  been  shown  by  one  investigator 
to  consist  of  a  mucoid  containing  12.7  per  cent  of  nitrogen  and  2.3 
per  cent  of  sulphur.  This  body  evidently  originates  in  the  urinary 
passages.  It  is  probably  slightly  soluble  in  the  urine.  Some  investigators 
believe  that  the  body  forming  the  nubecula  of  normal  urine  is  nucleo- 
protein  and  not  a  mucin  or  mucoid  as  stated  above.  A  discussion  of 
nucleoprotein  and  related  bodies  occurring  in  the  urine  under  pathological 
conditions  will  be  found  on  page  339. 

NH-CO 

OXALURIC  ACID,  CO      I 

NH,  CO  OH. 

Oxaluric  acid  is  not  a  constant  constituent  of  normal  human  urine, 
and  when  found  occurs  only  in  traces  as  the  ammonium  salt.  Upon 
boiling  oxaluric  acid  it  splits  into  oxalic  acid  and  urea. 


URINE.  309 

ENZYMES. 

Various  types  of  enzymes  produced  within  the  organism  are  excreted 
in  both  the  feces  and  the  urine.  In  this  connection  it  is  interesting  to 
note  that  pepsin,  gastric  rennin,  and  an  amylase  have  been  positively 
identified  in  the  urine.  The  occurrence  of  trypsin  in  the  urine,  at  least 
under  normal  conditions,  is  questioned. 

VOLATILE  FATTY  ACIDS. 

Acetic,  butyric,  and  formic  acids  have  been  found  under  normal 
conditions  in  the  urine  of  man  and  of  certain  carnivora  as  well  as  in  the 
urine  of  herbivora.  Normally  they  arise  principally  from  the  fermentation 
of  carbohydrates  and  the  putrefaction  of  proteins.  The  acids  containing 
the  fewest  carbon  atoms  (formic  and  acetic)  are  found  to  be  present  in 
larger  percentage  than  those  which  contain  a  larger  number  of  such 
atoms.  The  volatile  fatty  acids  occur  in  normal  urine  in  traces,  the 
total  output  for  twenty-four  hours,  according  to  different  investigators, 
varying  from  0.008  gram  to  0.05  gram. 

Pathologically,  the  excretion  of  volatile  fatty  acids  is  increased  in 
diabetes,  fevers,  and  in  certain  hepatic  diseases  in  which  the  parenchyma 
of  the  liver  is  seriously  affected.  Under  other  pathological  conditions  the 
output  may  be  diminished.  These  variations,  however,  in  the  excretion 
of  the  volatile  fatty  acids  possess  very  little  diagnostic  value. 

CH, 

I 
PARALACTIC  ACID,  CH(OH) 

COOH. 

Paralactic  acid  is  supposed  to  pass  into  the  urine  when  the  supply 
of  oxgyen  in  the  organism  is  diminished  through  any  cause,  e.  g.,  after 
acute  yellow  atrophy  of  the  liver,  acute  phosphorus  poisoning,  or  epi- 
leptic attacks.  This  acid  has  also  been  found  in  the  urine  of  healthy 
persons  following  the  physical  exercise  incident  to  prolonged  marching. 
Paralactic  acid  has  been  detected  in  the  urine  of  birds  after  the  removal 
of  the  liver.  Underbill  reports  the  occurrence  of  this  acid  in  the  urine 
of  a  case  of  pernicious  vomiting  of  pregnancy. 

CH2.CO.NH.CH2.COOH. 


PHENACETURIC  ACID, 

Phenaceturic  acid  occurs  principally  in   the  urine  of  herbivorous 
animals  but  has  frequently  been  detected  in  human  urine.     It  is  pro- 


[lO  PHYSIOLOGICAL    CHEMISTRY. 


duced  in  the  organism  through  the  synthesis  of  glycocoll  and  phenyl- 
acetic  acid.  It  may  be  decomposed  into  its  component  parts  by  boiling 
with  dilute  mineral  acids.  The  crystalline  form  of  phenaceturic  acid 
(small  rhombic  plates  with  rounded  angles)  resembles  one  form  of 
uric  acid  crystal. 


PHOSPHORIZED  COMPOUNDS. 


Phosphorus  in  organic  combination  has  been  found  in  the  urine 
in  such  bodies  as  glycerophosphoric  acid,  which  may  arise  from  the 
decomposition  of  lecithin,  and  phosphocarnic  acid.  It  is  claimed  that 
on  the  average  about  2.5  per  cent  of  the  total  phosphorus  elimination 
is  in  organic  combination. 


PIGMENTS. 

There  are  at  least  three  pigments  normally  present  in  human  urine. 
These  pigments  are  urochrome,  urobilin,  and  uroeryihrin. 

A.  UROCHROME. 

This  is  the  principal  pigment  of  normal  urine  and  imparts  the  char- 
acteristic yellow  color  to  that  fluid.  It  is  apparently  closely  related 
to  its  associated  pigment  urobilin  since  the  latter  may  be  readily  con- 
verted into  urochrome  through  evaporation  of  its  aqueous-ether  solution. 
Urochrome  may  be  obtained  in  the  form  of  a  brown,  amorphous 
powder  which  is  readily  soluble  in  water  and  95  per  cent  alcohol.  It 
is  less  soluble  in  absolute  alcohol,  acetone,  amyl  alcohol,  and  acetic 
ether  and  insoluble  in  benzene,  chloroform,  and  ether.  Urochrome  is 
said  to  be  a  nitrogenous  body  (4.2  per  cent  nitrogen),  free  from  iron. 

B.  UROBILIN. 

UrobiUn,  which  was  at  one  time  considered  to  be  the  principal  pig- 
ment of  urine,  in  reality  contributes  little  toward  the  pigmentation 
of  this  fluid.  It  is  claimed  that  no  urobilin  is  present  in  freshly  voided 
normal  urine  but  that  its  precursor,  a  chromogen  called  urobilinogen, 
is  present  and  gives  rise  to  urobilin  upon  decomposition  through  the 
influence  of  light.  It  is  claimed  by  some  investigators  that  there  are 
various  forms  of  urobihn,  e.  g.,  normal,  febrile,  physiological,  and  patho- 
logical. Urobilin  is  said  to  be  very  similar  to,  if  not  absolutely  identical 
with,  hydrobilirubin  (see  page  179). 

Urobilin  may  be  obtained  as  an  amorphous  powder  which  varies 
in  color  from  brown  to  reddish-brown,  red  and  reddish-yellow,  depend- 


URINE. 


3" 


ing  upon  the  way  in  which  it  is  prepared.  It  is  easily  soluble  in  ethyl 
alcohol,  amy!  alcohol,  and  chloroform,  and  slightly  soluble  in  ether, 
acetic  ether,  and  in  water.  Its  solutions  show  characteristic  absorption- 
bands  (see  Absorption  Spectra,  Plate  II).  Under  normal  conditions 
urobilin  is  derived  from  the  bile  pigments  in  the  intestine. 

Urv)bilin  is  increased  in  most  acute  infectious  diseases  such  as  ery- 
sipelas, malaria,  pneumonia,  and  scarlet  fever.  It  is  also  increased  in 
appendicitis,  carcifioma  of  the  liver,  catarrhal  icterus,  pernicions  ancemia, 
and  in  cases  of  poisoning  by  antifebrin,  antipyrin,  pyridin,  and  potas- 
sium chlorate.  In  general  it  is  usually  increased  when  blood  destruction 
is  excessive  and  in  many  disturbances  of  the  liver.  It  is  markedly 
decreased  in  phosphorus  poisoning. 

Experiments. 

1.  Spectroscopic  Examination.— Acidify  the  urine  with  hydro- 
chloric acid  and  allow  it  to  remain  exposed  to  the  air  for  a  few  moments. 
By  this  means  if  any  urobilinogen  is  present  it  will  be  transformed  into 
urobilin.  The  urine  may  now  be  examined  by  means  of  the  spectro- 
scope. If  urobiHn  is  present  in  the  fluid  the  characteristic  absorption- 
band  lying  between  b  and  F  will  be  observed  (see  Absorption  Spectra, 
Plate  II).  It  may  be  found  necessary  to  dilute  the  urine  with  water 
before  a  distinct  absorption-band  is  observed.  This  test  may  be  modi- 
fied by  acidifying  lo  c.c.  of  urine  with  hydrochloric  acid  and  shaking  it 
gently  with  5  c.c.  of  amyl  alcohol.  The  alcoholic  extract  when  examined 
spectroscopically  will  show  the  characteristic  urobilin  absorption-band. 
(Note  the  spectroscopic  examination  in  the  next  experiment.) 

2.  Ammoniacal-zinc  Chloride  Test. — Render  some  of  the  urine 
ammoniacal  by  the  addition  of  ammonium  hydroxide,  and  after  allowing 
it  to  stand  a  short  time  filter  off  the  precipitate  of  phosphates  and  add 
a  few  drops  of  zinc  chloride  solution  to  the  filtrate.  Observe  the  pro- 
duction of  a  greenish  fluorescence.  Examine  the  fluid  by  means  of  the 
spectroscope  and  note  the  absorption-band  which  occupies  much  the 
same  position  as  the  absorption-band  of  urobilin  in  acid  solution  (see 
Absorption  Spectra,  Plate  II). 

3.  Gerhardt's  Test. — To  20  c.c.  of  urine  add  3-5  c.c.  of  chloro- 
form and  shake  well.  Separate  the  chloroform  extract  and  add  to 
it  a  few  drops  of  iodine  solution  (I  in  KI).  Render  the  mixture  alka- 
line with  dilute  solution  of  potassium  hydroxide  and  note  the  produc- 
tion of  a  yellow  or  yellowish-brown  color.  The  solution  ordinarily 
exhibits  a  greenish  fluorescence. 

4.  Wirsing's  Test. — To   20  c.c.  of  urine  add  3-5  c.c.  of  chloro- 


312  PHYSIOLOGICAL   CHEMISTRY. 

form  and  shake  gently.  Separate  the  chloroform  ^  extract  and  add 
to  it  a  drop  of  an  alcoholic  solution  of  zinc  chloride.  Note  the  rose- 
red  color  and  the  greenish  fluorescence.  If  the  solution  is  turbid  it 
may  be  rendered  clear  by  the  addition  of  a  few  c.c.  of  absolute  alcohol. 

5,  Ether-Absolute  Alcohol  Test. — Mix  urine  and  pure  ether 
in  equal  volumes  and  shake  gently  in  a  separatory  funnel.  Separate 
the  ether  extract,  evaporate  it  to  dryness,  and  dissolve  the  residue  in 
2-3  c.c.  of  absolute  alcohol.  Note  the  greenish  fluorescence.  Examine 
the  solution  spectroscopically  and  observe  the  characteristic  absorp- 
tion-band (see  Absorption  Spectra,  Plate  II). 

6.  Ring  Test. — Acidify  25  c.c.  of  urine  with  2-3  drops  of  concen- 
trated hydrochloric  acid,  add  5  c.c.  of  chloroform  and  shake  the  mix- 
ture. Separate  the  chloroform,  place  it  in  a  test-tube,  and  add  care- 
fully 3-5  c.c.  of  an  alcoholic  solution  of  zinc  acetate.  Observe  the 
formation  of  a  green  ring  at  the  zone  of  contact  of  the  two  fluids.  If 
the  tube  is  shaken  a  fluorescence  may  be  observed. 

C.  UROERYTHRIN. 

This  pigment  is  frequently  present  in  small  amount  in  normal  urine. 
The  red  color  of  urinary  sediments  is  due  in  great  part  to  the  presence 
of  uroerythrin.  It  is  easily  soluble  in  amyl  alcohol,  slightly  soluble  in 
acetic  ether,  absolute  alcohol,  or  chloroform,  and  nearly  insoluble  in 
water.  Dilute  solutions  of  uroerythrin  are  pink  in  color  while  concen- 
trated solutions  are  orange-red  or  bright  red :  none  of  its  solutions  fluor- 
esce. Uroerythrin  is  increased  in  amount  after  strenuous  physical  exer- 
cise, digestive  disturbances,  fevers,  certain  liver  disorders,  and  in  various 
other  pathological  conditions. 

PTOMAINES  AND  LEUCOMAINES. 

These  toxic  substances  are  said  to  be  present  in  small  amount  in 
normal  urine.  Very  little  is  known,  definitely,  however,  about  them. 
It  is  claimed  that  five  different  poisons  may  be  detected  in  the  urine, 
and  it  is  further  stated  that  each  of  these  substances  produces  a  spe- 
cific and  definite  symptom  when  injected  intravenously  into  a  rabbit. 
The  resulting  symptoms  are  narcosis,  sahvation,  mydriasis,  paralysis, 
and  convulsions.  The  day  urine  is  principally  narcotic  and  is  2-4 
times  as  toxic  as  the  night  urine  which  is  chiefly  productive  of 
convulsions. 

PURINE  BASES. 
The  purine  bases  found  in  human  urine  are  adenine,  carnine,  epi- 
guanine,  episarkine,  guanine,  xanthine,  heteroxanthine,  hypoxanthine, 
paraxanthine,   and    i-methylxanthine.     The  main  bulk  of  the  purine 


URINE.  313 

base  content  of  the  urine  is  made  up  of  paraxanthine,  heteroxanthine 
and  i-methylxanthine  which  are  derived  for  the  most  part  from  the  caf- 
feine, theobromine,  and  theophylline  of  the  food.  The  total  purine  base 
content  is  made  up  of  the  products  of  two  distinct  forms  of  metabolism, 
f.  e.,  metabolism  of  ingested  nucleins  and  purines  and  metabolism  of 
tissue  nuclein  material.  Purine  bases  resulting  from  the  first  form  of 
metabolism  are  said  to  be  of  exogenous  origin  whereas  those  resulting  from 
the  second  form  of  metabolism  are  said  to  be  of  endogenous  origin.  The 
daily  output  of  purine  bases  by  the  urine  is  extremely  small  and  varies 
greatly  with  the  individual  (16-60  milligrams).  The  output  is  increased 
after  the  ingestion  of  nuclein  material  as  well  as  after  the  increased  destruc- 
tion of  leucocytes.  A  well  marked  increase  accompanies  leukaemia. 
Edsall  has  shown  that  the  output  of  purine  bases  by  the  urine  is  in- 
creased as  a  result  of  X-ray  treatment. 

Experiment. 

I.  Formation  of  the  Silver  Salts. — Add  an  excess  of  magnesia 
mixture^  to  25  c.c.  of  urine.  Filter  off  the  precipitate  and  add  am- 
moniacal  silver  solution"  to  the  filtrate.  A  precipitate  composed  of 
the  silver  salts  of  the  various  purine  bases  is  produced.  The  purine 
bases  may  be  determined  quantitatively  by  means  of  Kriiger  and  Schmidt's 
method  (see  p.  429),  or  Welker's  method  (see  p.  328). 

2.  Inorganic  Physiological  Constituents. 

Ammonia. 

Next  to  urea,  ammonia  is  the  most  important  of  the  nitrogenous 
end-products  of  protein  metabolism.  Ordinarily  about  2.5-4.5  per 
cent  of  the  total  nitrogen  of  the  urine  is  eliminated  as  ammonia  and 
on  the  average  this  would  be  about  0.7  gram  per  day.  Under  normal 
conditions  the  ammonia  is  present  in  the  urine  in  the  form  of  the  chloride, 
phosphate,  or  sidphate.  This  is  due  to  the  fact  that  combinations  of  this 
sort  are  not  oxidized  in  the  organism  to  form  urea,  but  are  excreted  as 
such.  This  explains  the  increase  in  the  output  of  ammonia  which  fol- 
lows the  administration  of  the  ammonium  salts  of  the  mineral  acids 
or  of  the  acids  themselves.  On  the  other  hand,  when  ammonium 
acetate  and  many  other  ammonium  salts  of  certain  organic  acids  are 
administered  no  increase  in  the  output  of  ammonia  occurs  since  the 
salt  is  oxidized  and  its  nitrogen  ultimately  appears  in  the  urine  as  urea. 

*  Magnesia  mixture  may  be  prepared  as  follows:  Dissolve  175  grams  of  MgS04  and 
350  grams  of  NH^Cl  in  1400  c.c.  of  distilled  water.  Add  700  grams  of  concentrated  NH^OH, 
mix  very  thoroughly  and  preserve  the  mixture  in  a  glass-stoppered  bottle. 

'  Ammoniacal  silver  solution  may  be  prepared  according  to  directions  given  on  page  430. 


314  PHYSIOLOGICAL   CHEMISTRY. 

Recent  experiments^  indicate  that  the  nitrogen  in  food  protein  may  in 
part  be  replaced  by  ammonium  salts. 

Copious  water  drinking  increases  the  ammonia  output.  This  fact  has 
been  interpreted  as  indicating  a  stimulation  of  the  gastric  secretion.^ 

The  acids  formed  during  the  process  of  protein  destruction  within 
the  body  have  an  influence  upon  the  excretion  of  ammonia  similar  to 
that  exerted  by  acids  which  have  been  administered.  Therefore  a 
pathological  increase  in  the  output  of  ammonia  is  observed  in  such 
diseases  as  are  accompanied  by  an  increased  and  imperfect  protein  me- 
tabolism, and  especially  in  diabetes,  in  which  disease  diacetic  acid  and 
/3-oxybutyric  acid  are  found  in  the  urine  in  combination  with  the 
ammonia. 

As  the  result  of  recent  experiments  Folin  claims  that  a  pronounced 
decrease  in  the  extent  of  protein  metabolism,  as  measured  by  the  total 
nitrogen  in  the  urine,  is  frequently  accompanied  by  a  decreased  elimi- 
nation of  ammonia.  The  ammonia  elimination  is  therefore  probably 
determined  by  other  factors  than  the  total  protein  catabolism  as  such. 
Furthermore,  he  believes  that  a  decided  decrease  in  the  total  nitrogen 
excretion  is  always  accompanied  by  a  relative  increase  in  the  ammonia- 
nitrogen,  provided  the  food  is  of  a  character  yielding  an  alkaline  ash. 

The  quantitative  determination  of  ammonia  must  be  made  upon 
the  fresh  urine  since  upon  standing  the  normal  urine  will  undergo  am- 
moniacal  fermentation  (see  page  276). 

Sulphates. 

Sulphur  in  combination  is  excreted  in  two  forms  in  the  urine;  first, 
as  loosely  combined,  unoxidized  or  neutral  sulphur,  and,  second,  as  oxidized 
or  acid  sulphur.  The  loosely  combined  sulphur  is  excreted  mainly  as 
a  constituent  of  such  bodies  as  cystine,  cysteine,  taurine,  hydrogen 
sulphide,  ethyl  sulphide,  thiocyanates,  sulphonic  acids,  oxyproteic  acid, 
alloxyproteic  acid,  and  uroferric  acid.  The  amount  of  loosely  com- 
bined sulphur  eliminated  is  in  great  measure  independent  of  the  extent 
of  protein  decomposition  or  of  the  total  sulphur  excretion.  In  this 
characteristic  it  is  somewhat  similar  to  the  excretion  of  creatinine.  The 
oxidized  sulphur  is  eliminated  in  the  form  of  sulphuric  acid,  principally 
as  salts  of  sodium,  potassium,  calcium,  and  magnesium;  a  relatively 
small  amount  occurs  in  the  form  of  ethereal  sulphuric  acid.  i.  e.,  sulphuric 
acid  in  combination  with  such  aromatic  bodies  as  phenol,  indole,  skatole, 
cresol,  pyrocatechin,  and  hydroquinone.     Sulphuric  acid  in  combination 

'  Grafe  and  Schlapfer:  Zeil.  physiol.  chem.,  77,  i,  1912,  experiments  by  Abderhalden  in 
same  journal. 

'Wills  and  Hawk:  Jaur.  Biol.  Chem.,  9,  xxx,  1911  (Proceedings). 


URINE.  315 

with  Na,  K,  Ca  or  Mg  is  sometimes  termqfl  inorganic  or  preformed 
sulphuric  acid,  whereas  the  ethereal  sulphuric  acid  is  sometimes  called 
cofijiigale  sulphuric  acid.  The  greater  part  of  the  sul[)hur  is  eliminated 
in  the  oxidized  form,  but  the  absolute  percentage  of  sulphur  excreted 
as  the  preformed,  ethereal  or  loosely  combined  type  depends  ujjon  the 
total  quantity  of  sulphur  present,  i.  e.,  there  is  no  definite  ratio  between 
the  three  forms  of  sulphur  which  will  apply  under  all  conditions.  The 
preformed  sulphuric  acid  may  be  precipitated  directly  from  acidified 
urine  with  BaClj,  whereas  the  ethereal  sulphuric  acid  must  undergo  a 
preliminary  boiling  in  the  presence  of  a  mineral  acid  before  it  can  be  so 
precipitated. 

The  sulphuric  acid  excreted  in  the  urine  arises  principally  from 
the  oxidation  of  protein  material  within  the  body;  a  relatively  small 
amount  is  due  to  ingested  sulphates.  Under  normal  conditions  about 
2.5  grams  of  sulphuric  acid  is  eliminated  daily.  Since  the  sulphuric 
acid  content  of  the  urine  has,  for  the  most  part,  a  protein  origin  and 
since  one  of  the  most  important  constituents  of  the  protein  molecule 
is  nitrogen,  it  would  be  reasonable  to  suppose  that  a  fairly  definite  ratio 
might  exist  between  the  excretion  of  these  two  elements.  However, 
when  we  appreciate  that  the  percentage  content  of  N  and  S  present  in 
different  proteins  is  subject  to  rather  wide  variations,  the  fixing  of  a  ratio 
which  will  express  the  exact  relation  existing  between  these  two  substances, 
as  they  appear  in  the  urine  as  end-products  of  protein  metabolism,  is 
practically  impossible.  It  has  been  suggested  that  the  ratio  5 :  i  expresses 
this  relation  in  a  general  way. 

Pathologically,  the  excretion  of  sulphuric  acid  by  the  urine  is  in- 
creased in  acute  fevers  and  in  all  other  diseases  marked  by  a  stimulated 
metabolism,  whereas  a  decrease  in  the  sulphuric  acid  excretion  is  observed 
in  those  diseases  which  are  accompanied  by  a  loss  of  appetite  and  a  dimin- 
ished metabolic  activity. 

Experiments. 

1.  Detection  of  Inorganic  Sulphuric  Acid.— Place  about  10  c.c. 
of  urine  in  a  test-tube,  acidify  with  acetic  acid  and  add  some  barium 
chloride  solution.     A  white  precipitate  of  barium  sulphate  forms. 

2.  Detection  of  Ethereal  Sulphuric  Acid. — Filter  off  the  barium 
sulphate  precipitate  formed  in  the  above  experiment,  add  i  c.c.  of  hydro- 
chloric acid  and  a  little  barium  chloride  solution  to  the  filtrate  and  heat 
the  mixture  to  boiling  for  1-2  minutes.  Note  the  appearance  of  a  tur- 
bidity due  to  the  presence  of  sulphuric  acid  which  has  been  separated 
from  the  ethereal  sulphates  and  has  combined  with  the  barium  of  the 
BaClj  to  form  BaSO^. 


;i6 


EEYSIOLOGICAL   CHEMISTRY. 


3.  Detection  of  Loosely  Combined  or  Neutral  Sulphur. — ^Place 
about  10  c.c.  of  urine  in  a  test-tube,  introduce  a  small  piece  of  zinc,  add 
sufficient  hydrochloric  acid  to  cause  a  gentle  evolution  of  hydrogen,  and 
over  the  mouth  of  the  tube  place  a  filter  paper  saturated  with  lead 
acetate  solution.     In  a  short  time  the  portion  of  the  paper  in  contact 

with  the  vapors  within  the  test-tube  be- 
comes blackened  due  to  the  formation 
of  lead  sulphide.  The  nascent  hydro- 
gen has  reacted  with  the  loosely  com- 
bined or  neutral  sulphur  to  form  hydro- 
gen sulphide  and  this  gas  coming  in 
contact  with  the  lead  acetate  paper 
has  caused  the  production  of  the  black 
lead  sulphide.  Sulphur  in  the  form  of 
inorganic  or  ethereal  sulphuric  acid  does 
not  respond  to  this  test. 

4,  Calcium  Sulphate  Crystals. — Place  10  c.c.  of  urine  in  a  test- 
tube,  add  10  drops  of  calcium  chloride  solution  and  allow  the  tube  to 
stand  until  crystals  form.  Examine  the  calcium  sulphate  crystals 
under  the  microscope  and  compare  them  with  those  shown  in  Fig.  100, 
above. 


Fig. 


100. — Calcium    Sulphate. 
{Hensel  and  Weil.) 


Chlorides. 

Next  to  urea,  the  chlorides  constitute  the  chief  solid  constituent 
of  the  urine.  The  principal  chlorides  found  in  the  urine  are  those  of 
sodium,  potassium,  ammonium,  and  magnesium,  with  sodium  chloride 
predominating.  The  excretion  of  chloride  is  dependent,  in  great  part, 
upon  the  nature  of  the  diet,  but  on  the  average  the  daily  output  is  about  10- 
15  grams,  expressed  as  sodium  chloride.  Copious  water-drinking  in- 
creases the  output  of  chlorides  considerably.  Because  of  their  solubility, 
chlorides  are  never  found  in  the  urinary  sediment. 

Since  the  amount  of  chlorides  excreted  in  the  urine  is  due  primarily 
to  the  chloride  content  of  the  food  ingested,  it  follows  that  a  decrease 
in  the  amount  of  ingested  chloride  will  likewise  cause  a  decrease  in  the 
chloride  content  of  the  urine.  In  cases  of  actual  fasting  the  chloride 
content  of  the  urine  may  be  decreased  to  a  slight  trace  which  is  derived 
from  the  body  fluids  and  tissues.  Under  these  conditions,  however, 
an  examination  of  the  blood  of  the  fasting  subject  will  show  the  per- 
centage of  chlorides  in  this  fluid  to  be  approximately  normal.  This 
forms  a  very  striking  example  of  the  care  nature  takes  to  maintain  the 
normal  composition  of  the  blood.     There  is  a  limit  to  the  power  of  the 


URINE.  317 

body  to  maintain  this  equilibrium,  however,  and  if  the  fasting  organism 
be  subjected  to  the  influence  of  diuretics  for  a  time,  a  point  is  reached 
where  the  composition  of  the  blood  can  no  longer  be  maintained  and  a 
gradual  decrease  in  its  chloride  content  occurs  which  finally  results  in 
death.  Death  is  supposed  to  result  not  so  much  because  of  a  lack  of 
chlorine  as  from  a  deficiency  of  sodium.  This  is  shown  from  the  fact  that 
potassium  chloride,  for  instance,  cannot  replace  the  sodium  chloride 
of  the  blood  when  the  latter  is  decreased  in  the  manner  above  stated. 
When  this  substitution  is  attempted  the  potassium  salt  is  excreted  at 
once  in  the  urine,  and  death  follows  as  above  indicated. 

Pathologically,  the  excretion  of  chlorides  may  be  decreased  in  some 
fevers,  chronic  nephritis,  croupous  pneumonia,  diarrhoea,  certain  stomach 
disorders,  and  in  acute  articular  rheumatism. 

Experiment. 

Detection  of  Chlorides  in  Urine. — Place  about  5  c.c.  of  urine  in 
a  test-tube,  render  it  acid  with  nitric  acid  and  add  a  few  drops  of  a 
solution  of  silver  nitrate.  A  white  precipitate,  due  to  the  formation 
of  silver  chloride,  is  produced.  This  precipitate  is  soluble  in  am- 
monium hydroxide. 

Phosphates. 

Phosphoric  acid  exists  in  the  urine  in  two  general  forms:  First, 
that  in  combination  with  the  alkali  metals,  sodium  and  potassium, 
and  the  radical  ammonium;  second,  that  in  combination  with  the  alkaline 
earths,  calcium  and  magnesium.  Phosphates  formed  through  a  union 
of  phosphoric  acid  with  the  alkali  metals  are  termed  alkaline  phosphates, 
or  phosphates  of  the  alkali  metals,  whereas  phosphates  formed  through 
a  union  of  phosphoric  acid  with  the  alkaline  earths  are  termed  earthy 
phosphates,  or  phosphates  of  the  alkaline  earths. 

Three  series  of  salts  are  formed  by  phosphoric  acid :  Normal,  MgPO^,  ^ 
mano-hydrogen,  M.HPO^,  and  di-hydrogen,  MH,PO^.  The  di-hydrogen 
salts  are  acid  in  reaction,  and  it  was  generally  believed  that  about  60 
per  cent  of  the  total  phosphate  content  of  the  urine  was  in  the  form  of 
this  type  of  salt,  and  that  the  acidity  of  the  urine  was  due  in  great  part  to 
the  presence  of  sodium  di-hydrogen  phosphate.  Recently,  however,  it 
has  been  quite  clearly  shown  that  the  normal  acidity  of  the  urine  is  not 
due  to  the  presence  of  this  salt,  but  is  due,  at  least  in  part,  to  the  presence 
of  various  acidic  radicals.  In  this  connection  Folin  believes  that  the 
phosphates  in  clear  acid  urine  are  all  of  the  mono-hydrogen  type,  and  that 
the  acidity  of  the  urines  of  this  character  is  generally  greater  than  the 

*  M  may  be  occupied  by  any  of  the  alkali  metals  or  alkaline  earths. 


3l8  PHYSIOLOGICAL    CHEMISTRY. 

combined  acidity  of  all  the  phosphates  present;  the  excess  in  the  acidity 
above  that  due  to  phosphates  be  beleives  to  be  due  to  free  organic  acids. 
Henderson'  maintains  that  "determinations  of  hydrogen  ionization  in 
urine  and  its  behavior  toward  indicators  both  support  the  view  that  in 
urine  there  exists  a  mixture  of  m&no-  and  di-hydrogen  phosphates  of 
sodium,  ammonium  and  other  bases."  The  observation  has  recently 
been  made  that  urine  may  be  separated  into  two  portions,  one  part  con- 
sisting almost  entirely  of  inorganic  matter  including  practically  all  of 
the  phosphates  and  having  an  alkaline  reaction,  the  other  containing 
practically  all  of  the  organic  substances  and  no  phosphates  and  having  an 
acid  reactian. 

In  bones  the  phosphates  occur  principally  in  the  form  of  the  normal 
salts  of  calcium  and  magnesium.  The  mono-hydrogen  salts  as  a  class 
are  alkaline  in  reaction  to  litmus,  and  it  is  to  the  presence  of  di-sodium 
hydrogen  phosphate,  Na2HP04,  that  the  greater  part  of  the  alkalinity 
of  the  saliva  is  due. 

The  excretion  of  phosphoric  acid  is  extremely  variable,  but  on  the 
average  the  total  output  for  24  hours  is  about  2.5  grams,  expressed 
as  P2O5.  Ordinarily  the  total  output  is  distributed  between  alkaline 
phosphates  and  earthy  phosphates  approximately  in  the  ratio  2:1.  The 
greater  part  of  this  phosphoric  acid  arises  from  the  ingested  food,  either 
from  the  preformed  phosphates  or  more  especially  from  the  phosphorus 
in  organic  combination  such  as  we  find  it  in  phospho-proteins,  nucleo- 
proteins  and  lecithins;  the  phosphorus-containing  tissues  of  the  body 
also  contribute  to  the  total  output  of  this  element.  Alkaline  phosphates 
ingested  with  the  food  have  a  tendency  to  increase  the  phosphoric  acid 
content  of  the  urine  to  a  greater  extent  than  the  earthy  phosphates  so 
ingested.  This  is  due,  in  a  measure,  to  the  fact  that  a  portion  of  the  earthy 
phosphates,  under  certain  conditions,  may  be  precipitated  in  the  intestine 
and  excreted  in  the  feces;  this  is  especially  to  be  noted  in  the  case  of 
herbivorous  animals.  Since  the  extent  to  which  the  phosphates  are 
absorbed  in  the  intestine  depends  upon  the  form  in  which  they  are  present 
in  the  food,  under  ordinary  conditions,  there  can  be  no  absolute  relation- 
ship between  the  urinary  output  of  nitrogen  and  phosphorus.  If  the 
diet  is  constant,  however,  from  day  to  day,  thus  allowing  of  the  prepara- 
tion of  both  a  nitrogen  and  a  phosphorus  balance,^  a  definite  ratio  may 
be  established.  In  experiments  upon  dogs,  which  were  fed  an  exclusive 
meat  diet,  the  ratio  of  nitrogen  to  phosphorus,  in  the  urine  and  feces, 
was  found  to  be  8.1 :  i. 

'  Henderson:  Am.  Jour.  Physiol.,  15,  257,  1906. 

2  In  metabolism  experiments,  a  statement  showing  the  relation  existing  between  the  nitro- 
gen content  of  the  food  on  the  one  hand  and  that  of  the  urine  and  feces  on  the  other,  for  a 
definite  period,  is  termed  a  nitrogen  balance  or  a  "  balance  of  the  income  and  outgo  of  nitrogen." 


URINE. 


319 


It  has  been  demonstrated  by  recent  investigation  that  the  ingestion  of 
inorganic  phosphorus  compounds  may  give  rise  to  organic  phosphorus 
compounds  such  as  lecithin,  phosphatides,  nucleoproteins  and  phospho- 
proteins.  This  is  an  instance  of  an  organic  substance  synthesized  from 
an  inorganic  substance.  The  experiments  have  been  made  principally 
on  ducks*  and  hens.' 

Pathologically  the  excretion  of  phosphoric  acid  is  increased  in  such 
diseases  of  the  bones  as  difluse  periostosis,  osteomalacia,  and  rickets; 
according  to  some  investigators,  in  the  early  stages  of  pulmonary  tuber- 
culosis, in  acute  yellow  atrophy  of  the  liver,  in  diseases  which  are  accom- 
panied by  an  extensive  decomposition  of  nervous  tissue,  and  after  sleep 
induced  by  potassium  bromide  or  chloral  hydrate  (Mendel).  It  is  also 
increased  after  copious  water-drinking.  A  decrease  in  the  excretion  of 
phosphates  is  at  times  noted  in  febrile  aflfections,  such  as  the  acute  infec- 
tious diseases;  in  pregnancy,  in  the  period  during  which  the  foetal  bones 
are  forming,  and  in  diseases  of  the  kidneys,  because  of  non-elimination. 

Experiments. 

I.  Formation  of  "Triple  Phosphate." — Place  some  urine  in  a 
beaker,   render  it   alkaline   with  ammoniu   mhydroxide,   add   a   small 


^ 


\ 


Fig.  ioi. — "Triple  Phosphate."     (Ogden.) 

amount  of  magnesium  sulphate  solution  and  allow  the  beaker  to  stand 
in  a  cool  place  over  night.  Q-ystals  of  ammonium  magnesium  phosphate, 
'^  triple  phosphate,^'  form  under  these  conditions.  Examine  the  crystalline 
sediment  under  the  microscope  and  compare  the  forms  of  the  crystals 
with  those  shown  in  Fig.  loi,  above. 

2.  "Triple  Phosphate"  Crystals  in  Ammoniacal  Fermentation. 
— Stand  some  urine  aside  in  a  beaker  for  several  days.     Ammoniacal 

*  Fingerling:  Biochem.  Zeit.,  38,  448,  IQ12. 

^McCollum  and  Halpin:  Jour.  Biol.  Client.,  11,  47  (Proceedings),  1912. 


320  PHYSIOLOGICAL   CHEMISTRY. 

fermentation  will  develop  and  "triple  phosphate"  crystals  will  form. 
Examine  the  sediment  under  the  microscope  and  compare  the  crystals 
with  those  shown  in  Fig.  loi,  below. 

3.  Detection  of  Earthy  Phosphates. — ^Place  10  c.c.  of  urine  in  a 
test-tube  and  render  it  alkaline  with  ammonium  hydroxide.  Warm 
the  mixture  and  note  the  separation  of  a  precipitate  of  earthy  phosphates. 

4.  Detection  of  Alkaline  Phosphates. — Filter  off  the  earthy 
phosphates  as  formed  in  the  last  experiment,  and  add  a  small  amount 
of  ^magnesia  mixture  (see  page  313)  to  the  filtrate.  Now  warm  the 
mixture  and  observe  the  formation  of  a  white  precipitate  due  to  the 
presence  of  alkaline  phosphates.  Note  the  difference  in  the  size  of 
the  precipitates  of  the  two  forms  of  phosphates  from  this  same  volume 
of  urine.  Which  form  of  phosphates  was  present  in  the  larger  amount, 
earthy  or  alkaline? 

5.  Influence  upon  Fehling's  Solution. — Place  2  c.c.  of  Fehling's 
solution  in  a  test-tube,  dilute  it  with  4  volumes  of  water  and  heat  to 
boiling.  Add  a  solution  of  sodium  dihydrogen  phosphate,  NaHgPO^, 
a  small  amount  at  a  time,  and  heat  after  each  addition.  What  do  you 
observe?  What  does  this  observation  force  you  to  conclude  regarding 
the  interference  of  phosphates  in  the  testing  of  diabetic  urine  by  means 
of  Fehling's  test? 

Sodium  and  Potassium. 

The  elements  sodium  and  potassium  are  always  present  in  the  urine. 
Usually  they  are  combined  with  such  acidic  radicals  as  CI,  CO3,  SO^ 
and.  PO4.  The  amount  of  potassium,  expressed  as  KgO,  excreted  in 
24  hours  by  an  adult,  subsisting  upon  a  mixed  diet,  is  on  the  average 
2-3  grams,  whereas  the  amount  of  sodium,  expressed  as  NajO,  under 
the  same  conditions,  is  ordinarily  4-6  grams.  The  ratio  of  K  to  Na  is 
generally  about  3:5.  The  absolute  quantity  of  these  elements  excreted 
depends,  of  course,  in  large  measure,  upon  the  nature  of  the  diet.  Because 
of  the  non-ingestion  of  NaCl  and  the  accompanying  destruction  of 
potassium-containing  body  tissues,  the  urine  during  fasting  contains  more 
potassium  salts  than  sodium  salts. 

Pathologically  the  output  of  potassium,  in  its  relation  to  sodium,  may 
be  increased  during  fever;  following  the  crisis,  however,  the  output  of  this 
element  may  be  decreased.  It  may  also  be  increased  in  conditions 
associated  with  acid  intoxication. 

Calcium  and  Magnesium. 

The  greater  part  of  the  calcium  and  magnesium  excreted  in  the 
urine  is  in  the  form  of  phosphates.     The  daily  output,  which  depends 


URINE.  321 

principally  upon  the  nature  of  the  diet,  aj^^regates  on  the  averaj^e  about 
I  gram  and  is  made  up  of  the  phosphates  of  calcium  and  magnesium 
in  the  proportion  of  1:2.  The  percentage  of  calcium  salts  present  in 
the  urine  at  any  one  time  forms  no  dependable  index  as  to  the  absorp- 
tion of  this  class  of  salts,  since  they  are  again  excreted  into  the  intestine 
after  absorption.  It  is  therefore  impossible  to  draw  any  satisfactory 
conclusions  regarding  the  excretion  of  the  alkaline  earths  unless  we 
obtain  accurate  analytical  data  from  both  the  feces  and  the  urine. 

V^ry  little  is  known  positively  regarding  the  actual  course  of  the 
excretion  of  the  alkaline  earths  under  pathological  conditions  except 
that  an  excess  of  calcium  is  found  in  acid  intoxication  and  some  diseases 
of  the  bones. 

Carbonates. 

Carbonates  generally  occur  in  small  amount  in  the  urine  of  man 
and  carnivora  under  normal  conditions,  whereas  much  larger  quanti- 
ties are  ordinarily  present  in  the  urine  of  herbivora.  The  alkaline 
reaction  of  the  urine  of  herbivora  is  dependable  in  great  measure  upon 
the  presence  of  carbonates.  In  general  a  urine  containing  carbonates 
in  appreciable  amount  is  turbid  when  passed  or  becomes  so  shortly 
after.  These  bodies  ordinarily  occur  as  alkali  or  alkaline  earth  com- 
pounds and  the  turbid  character  of  urine  containing  them  is  usually 
due  principally  to  the  latter  class  of  substances.  The  carbonates  of 
the  alkaline  earths  are  often  found  in  amorphous  urinary  sediments. 

Iron. 

Iron  is  present  in  small  amount  in  normal  urine.  It  probably  occurs 
partly  in  inorganic  and  partly  in  organic  combination.  The  iron  con- 
tained in  urinary  pigments  or  chromogens  is  in  organic  combination. 
According  to  different  investigators  the  iron  content  of  normal  urine  will 
probably  not  average  more  than  0.00 1  gram  per  day. 

Experiment. 

Detection  of  Iron  in  Urine. — Evaporate  a  convenient  volume 
(10-15  c.c.)  of  urine  to  dryness.  Incinerate  and  dissolve  the  residue 
in  a  few  drops  of  iron-free  hydrochloric  acid  and  dilute  the  acid  solu- 
tion with  5  c.c.  of  water.  Divide  the  acid  solution  into  two  parts  and 
make  the  following  tests:  (a)  To  the  first  part  add  a  solution  of  ammo- 
nium thiocyanate;  a  red  color  indicates  the  presence  of  iron,  {b)  To 
the  second  part  of  the  solution  add  a  little  potassium  ferrocyanide  solu- 
tion; aprecipitate  of  Prussian  blue  forms  upon  standing. 


322  PHYSIOLOGICAL   CHEMISTRY, 

Fluorides,  Nitrates,  Silicates  and  Hydrogen  Peroxide. 

These  substances  are  all  found  in  traces  in  human  urine  under 
normal  conditions.  Nitrates  are  undoubtedly  introduced  into  the 
organism  in  the  water  and  ingested  food.  The  average  excretion  of 
nitrates  is  about  0.5  gram  per  day,  the  output  being  the  largest  upon  a 
vegetable  diet  and  smallest  upon  a  meat  diet.  Nitrites  are  found  only 
in  urine  which  is  undergoing  decomposition  and  are  formed  from 
nitrates  in  the  course  of  ammoniacal  fermentation.  Hydrogen  peroxide 
has  been  detected  in  the  urine,  but  its  presence  is  believed  to  possess_^no 
pathological  importance. 


CHAPTER  XIX. 
URINE:  PATHOLOGICAL  CONSTITUENTS.^ 


Dextrese. 


Proteins 


Serum  albumin. 
Serum  globulin. 

f  Dcutero-proteose. 
Proteoses  |  Hetero-proteose. 

"Bence- Jones'  protein." 


Peptone. 

Nucleoprotein. 

Fibrin. 

Oxyhaemoglobin. 

^,      ,     [  Form  elements. 
Blood     <^  ^. 

[  Pigment. 

Bile       f  ^'S™'"'^- 
1  Acids. 

Creatine.^ 

Acetone. 

Diacetic  acid. 

,5-Oxybutyric  acid. 

Conjugate  glycuronates. 

Pentoses. 

Fat. 

Haema  toporphy  rin . 

Lactose. 

Galactose. 

Laevulose. 

Inosite. 

Laiose. 

Melanin. 

L'rorosein. 

Unknown  substances. 

DEXTROSE. 

Traces  of  this  sugar  occur  in  normal  urine,  but  the  amount  is  not 
sufficient  to   be    readily  detected   by  the    ordinary  simple    qualitative 

*  See  note  at  the  bottom  of  page  283. 

*  Normal  constituent  of  urine  of  infants  and  children. 


324  PHYSIOLOGICAL   CHEMISTRY. 

tests.  There  are  two  distinct  types  of  pathological  glycosuria,  i.  e., 
transitory  glycosuria  and  persistent  glycosuria.  The  transitory  type 
may  follow  the  ingestion  of  an  excess  of  sugar,  causing  the  assimilation 
limit^  to  be  exceeded,  or  it  may  accompany  any  one  of  several  disorders 
which  cause  impairment  of  the  power  of  assimilating  sugar.  In  the 
persistent  type  large  amounts  of  sugar  are  excreted  daily  in  the  urine 
for  long  periods  of  time.  Under  such  circumstances  a  condition  known 
as  diabetes  mellitus  exists.  Ordinarily,  diabetic  urine  which  contains 
•a  Jiigh  percentage  of  sugar  possesses  a  faint  yellow  color,  a  high  specific 
gravity,  and  a  volume  which  is  above  normal. 

Experiments. 

I.  Phenylhydrazine  Reaction. — Test  the  urine  according  to  one 
of  the-  following  methods:  {a)  To  a  small  amount  of  phenylhydrazine 
mixture,  furnished  by  the  instructor,^  add  5  c.c.  of  the  urine,  shake 
well,  and  heat  on  a  boiling  water-bath  for  one-half  to  three-quarters  of 
an  hour.  Allow  the  tube  to  cool  slowly  and  examine  the  crystals  micro- 
scopically (Plate  III,  opposite  page  28).  If  the  solution  has  become 
too  concentrated  in  the  boiling  process  it  will  be  light-red  in  color  and 
no  crystals  will  separate  until  it  is  diluted  with  water. 

Yellow  crystalline  bodies  called  osazones  are  formed  from  certain 
sugars  under  these  conditions,  in  general  each  individual  sugar  giving 
rise  to  an  osazone  of  a  definite  crystalline  form  which  is  typical  for  that 
sugar.  It  is  important  to  remember  in  this  connection  that,  of  the 
simple  sugars  of  interest  in  physiological  chemistry,  dextrose  and  laevu- 
lose  yield  the  same  osazone,  with  phenylhydrazine.  Each  osazone  has 
a  definite  melting-point,  and  as  a  further  and  more  accurate  means  of 
identification  it  may  be  recrystallized  and  identified  by  the  determina- 
tion of  its  melting-point  and  nitrogen  content.  The  reaction  taking 
place  in  the  formation  of  phenyldextrosazone  is  as  follows : 

C„H,,0„  +  2(H3N.NH.C«HJ-^CeH,,0,(N.NH.CeH3)3+2H30-fH,. 

Dextrose.  Phenylhydrazine.  Phenyldextrosazone. 

(b)  Place  5  c.c.  of  the  urine  in  a  test-tube,  add  i  c.c.  of  phenylhy- 
drazine-acetate  solution  furnished  by  the  instructor,^  and  heat  on  a 
boiling  water-bath  for  one-half  to  three-quarters  of  an  hour.  Allow 
the  liquid  to  cool  slowly  and  examine  the  crystals  microscopically  (Plate 
III,  opposite  p.  28). 

'The  assimilation  limit  for  dextrose  has  been  shown  to  be  between  loo  and  150  grams 
(Brasch:  Zeit.filr  Biol.,  50,  113,  1907. 

■■'  This  mixture  is  prepared  by  combining  one  part  of  phenylhydrazine-hydrochloride  and 
two  parts  of  sodium  acetate,  by  weight.     These  are  thoroughly  mixed  in  a  mortar. 

'  This  solution  is  prepared  by  mixing  one  part  by  volume,  in  each  case,  of  glacial  acetic 
acid,  one  part  of  water  and  two  parts  of  phenylhydrazine  (the  base). 


URINE.  325 

The  phenyl  hydrazine  test  has  been  so  modified  by  Cipollina  as  to 
be  of  use  as  a  rapid  clinical  test.  The  directions  for  this  test  are  given 
in  the  next  experiment. 

2.  Cipollina's  Test. — Thoroughly  mix  4  c.c.  of  urine,  5  drops  of 
phenylhydrazine  (the  base)  and  1/2  c.c.  of  glacial  acetic  acid  in 
a  test-tube.  Heat  the  mixture  for  about  one  minute  over  a  low  flame, 
shaking  the  tube  continually  to  prevent  loss  of  fluid  by  bumping.  Add 
4-5  drops  of  potassium  hydroxide  or  sodium  hydroxide  (sp.  gr.  1.16), 
being  certain  that  the  fluid  in  the  test-tube  remains  acid;  heat  the  mix- 
ture again  for  a  moment  and  then  cool  the  contents  of  the  tube.  Ordi- 
narily the  crystals  form  at  once,  especially  if  the  urine  possesses  a  low 
specific  gravity.  If  they  do  not  appear  immediately  allow  the  tube  to 
stand  at  least  20  minutes  before  deciding  upon  the  absence  of  sugar. 

Examine  the  crystals  under  the  micrscope  and  compare  them  with 
those  shown  in  Plate  III,  opposite  page  28. 

3.  Riegler's  Reaction.^ — Introduce  o.i  gram  of  phenylhydrazine- 
hydrochloride  and  0.25  gram  of  sodium  acetate  into  a  test-tube,  add 
20  drops  of  the  urine  under  examination,  and  heat  the  mixture  to  boiling. 
Now  introduce  10  c.c.  of  a  3  per  cent  solution  of  potassium  hydroxide 
and  gently  shake  the  tube  and  contents.  If  the  urine  under  examination 
contains  dextrose  the  liquid  in  the  tube  will  assume  a  red  color.  One 
per  cent  dextrose  yields  an  immediate  color  whereas  0.05  per  cent  yields 
the  color  only  after  the  lapse  of  a  period  of  one-half  hour  from  the  time 
the  alkali  is  added.  If  the  color  appears  after  the  30-minute  interval 
the  color  change  is  without  significance  inasmuch  as  sugar-free  urines 
will  respond  thus.  The  reaction  is  given  by  all  aldehydes  and  therefore 
the  test  cannot  be  safely  employed  in  testing  urines  preserved  by  formalde- 
hyde.    Albumin  does  not  interfere  with  the  test. 

4.  Bottu's  Test.- — To  8  c.c.  of  Bottu's  reagent^  in  a  test-tube  add 
I  c.c.  of  the  urine  under  examination  and  mix  the  liquids  by  gentle 
shaking.  Now  heat  the  upper  portion  of  the  mixture  to  boiling,  add 
an  additional  i  c.c.  of  urine  and  heat  the  mixture  again  immediately. 
The  appearance  of  a  blue  color  accompanied  by  the  precipitation  of 
small  particles  of  indigo  blue  indicates  the  presence  of  dextrose  in  the 
urine  under  examination.  The  test  will  serve  to  detect  the  presence 
of  0.1  per  cent  of  dextrose  and  is  uninfluenced  by  creatinine  or  by 
ammonium  salts. 

5.  Reduction  Tests. — To  their  aldehyde  or  ketone  structure  many 
sugars  owe  the  property  of  readily  reducing  the  alkaline  solutions  of  the 

'  Riegler:  Compt.  rend.  soc.  biol.,  66,  p.  795. 
-  Bottu:  Cumpt.  rend.  soc.  biol.,  66,  p.  972. 

^  This  reagent  contains  3.5  grams  of  o-nitrophenylpropiolic  acid  and  5  c.c.  of  a  freshly  pre- 
pared ID  per  cent  solution  of  sodium  hydro.xide  per  liter. 


326  PHYSIOLOGICAL   CHEMISTRY. 

oxides  of  metals  like  copper,  bismuth,  and  mercury;  they  also  possess 
the  property  of  reducing  ammoniacal  silver  solutions  with  the  separa- 
tion of  metallic  silver.  Upon  this  property  of  reduction  the  most  widely 
used  tests  for  sugars  are  based.  When  whitish-blue  cupric  hydroxide  in 
suspension  in  an  alkaline  liquid  is  heated  it  is  converted  into  insoluble 
black  cupric  oxide,  but  if  a  reducing  agent  like  certain  sugars  be  present 
the  cupric  hydroxide  is  reduced  to  insoluble  yellow  cuprous  hydroxide, 
which  in  turn  on  further  heating  may  be  converted  into  brownish-red  or 
red  cuprous  oxide.     These  changes  are  indicated  as  follows: 

OH 

/ 
Cu         —     Cu^O  +  H^O. 

\  Cupric  oxide 

(black). 

OH 

Cupric  hydroxide 
(whitish-blue) . 

I 

OH 

/ 

Cu 

\ 
OH 

-^  2Cu-0H+H,0-^0. 

OTT       Cuprous  hydroxide 
^"-  (yellow). 

Cu 
\ 
\ 
OH 

Cu 

Cu— OH        \ 
I  -^       O+H^O. 

Cu— OH        / 
Cu 

Cuprous  hydroxide      Cuprous  oxide 
(yellow).  (brownish- red) . 

The  chemical  equations  here  discussed  are  exemplified  in  Trommer's 
and  Fehling's  tests. 

(a)  Trommer's  Test. — To  5  c.c.  of  urine  in  a  test-tube  add  one- 
half  its  volume  of  KOH  or  NaOH.  Mix  thoroughly  and  add,  drop  by 
drop,  agitating  after  the  addition  of  each  drop,  a  very  dilute  solution  of 
copper  sulphate.  Continue  the  addition  until  there  is  a  slight  perma- 
nent precipitate  of  cupric  hydroxide  and  in  consequence  the  solution  is 
slightly  turbid.  Heat,  and  the  cupric  hydroxide  is  reduced  to  yellow 
cuprous  hydroxide  or  to  brownish-red  cuprous  oxide.     If  the  solution 


URINE.  327 

of  copper  sulphate  used  is  too  strong,  a  small  brownish-red  precipitate 
produced  in  the  presence  of  a  low  percentage  of  dextrose  may  be  entirely 
masked.  On  the  other  hand,  if  too  little  copper  sulphate  is  used  a  light- 
colored  precipitate  formed  by  uric  acid  and  purine  bases  may  obscure 
the  brownish-red  precipitate  of  cuprous  oxide.  The  action  of  KOH 
or  NaOH  in  the  presence  of  an  excess  of  sugar  and  insufl5cient  copper 
will  produce  a  brownish  color.  Phosphates  of  the  alkaline  earths  may 
also  be  precipitated  in  the  alkaline  solution  and  be  mistaken  for  cuprous 
hydroxide.     Trommer's  test  is  not  very  satisfactory. 

Salkowski^  has  very  recently  proposed  a  modification  of  the  Trom- 
mer  procedure  which  he  claims  is  a  very  accurate  sugar  test. 

(b)  Fehling's  Test. — To  about  i  c.c.  of  Fchling's  solution"  in  a  test- 
tube  add  about  4  c.c.  of  water,  and  boil.  This  is  done  to  determine 
whether  the  solution  will  of  itself  cause  the  formation  of  a  precipitate 
of  brownish-red  cuprous  oxide.  If  such  a  precipitate  forms,  the  Feh- 
ling's  solution  must  not  be  used.  Add  urine  to  the  warm  Fehling's 
solution,  a  few  drops  at  a  time,  and  heat  the  mixture  after  each  addition. 
The  production  of  yellow  cuprous  hydroxide  or  brownish-red  cuprous 
oxide  indicates  that  reduction  has  taken  place.  The  yellow  precipi- 
tate is  more  likely  to  occur  if  the  urine  is  added  rapidly  and  in  large 
amount,  whereas  with  a  less  rapid  addition  of  smaller  amounts  of  urine 
the  brownish-red  precipitate  is  generally  formed. 

This  is  a  much  more  satisfactory  test  than  Trommer's,  but  even 
this  test  is  not  entirely  reliable  when  used  to  detect  sugar  in  the  urine. 
Such  bodies  as  c&njugate  glycuronaies,  uric  acid,  nucleoprotein,  and  honio- 
gentisic  acid,  when  present  in  sufficient  amount,  may  produce  a  result 
similar  to  that  produced  by  sugar.  Phosphates  of  the  alkaline  earths 
may  be  precipitated  by  the  alkali  of  the  Fehling's  solution  and  in  appear- 
ance may  be  mistaken  for  the  cuprous  hydroxide.  Cupric  hydroxide 
may  also  be  reduced  to  cuprous  oxide  and  this  in  turn  be  dissolved  by 
creatinine,  a  normal  urinary  constituent.  This  will  give  the  urine  under 
examination  a  greenish  tinge  and  may  obscure  the  sugar  reaction  even 
when  a  considerable  amount  of  sugar  is  present.  According  to  Laird^ 
even  small  amounts  of  creatinine  will  retard  the  reaction  velocity  of  re- 
ducing sugars  with  Fehling's  solution. 

'  Salkowski:  Zeit.  physiol.  Chem.,  79,  164,  1912. 

^  Fehling's  solution  is  composed  of  two  definite  solutions — a  copper  sulphate  solution  and 
an  alkaline  tartrate  solution,  which  may  be  prepared  as  follows: 

Copper  sulphate  solution  =  24.6^  grams  of  copper  sulphate  dissolved  in  water  and  made 
up  to  500  c.c. 

Alkaline  tartrate  solution  — 12$  grams  of  potassium  hydroxide  and  173  grams  of  Rochelle 
salt  dissolved  in  water  and  made  up  to  500  c.c. 

These  solutions  should  be  preserved  separately  in  rubber-stoppered  bottles  and  mixed 
in  equal  volumes  when  needed  for  use.     This  is  done  to  prevent  deterioration. 

'Laird:  Journ.  Path,  and  Bad.,  16,  398,  1912. 


328  PHYSIOLOGICAL   CHEMISTRY. 

(c)  Benedict's  Modifications  of  Fehling's  Test.  First  Modification. — 
To  2  c.c.  of  Benedict's  solution^  in  a  test-tube  add  6  c.c.  of  distilled 
water  and  7-9  drops  (not  more)  of  the  urine  under  examination.  Boil 
the  mixture  vigorously  for  about  15-30  seconds  and  permit  it  to  cool 
to  room  temperature  spontaneously.  (If  desired  this  process  may  be 
repeated,  although  it  is  ordinarily  unnecessary.)  If  sugar  is  present 
in  the  solution  a  precipitate  will  form  which  is  often  bluish-green  or 
green  at  first,  especially  if  the  percentage  of  sugar  is  low,  and  which 
usually  becomes  yellowish  upon  standing.  If  the  sugar  present  exceeds 
0.06  per  cent  this  precipitate  generally  forms  at  or  below  the  boiling- 
point,  whereas  if  less  than  0.06  per  cent  of  sugar  is  present  the  precipi- 
tate forms  more  slowly  and  generally  only  after  the  solution  has  cooled. 
The  greenish  precipitate  obtained  with  urines  containing  small  amounts 
of  sugar  may  be  a  compound  of  copper  with  the  sugar  or  a  compound 
of  some  constituent  of  the  urine  with  reduced  copper  oxide  instead  of 
being  a  precipitate  of  cuprous  hydroxide  or  oxide  as  is  the  case  when 
the  original  Fehling  solution  is  reduced. 

Benedict  claims  that,  whereas  the  original  Fehling's  test  will  not 
serve  to  detect  sugar  when  present  in  a  concentration  of  less  than  o.  i  per 
cent,  that  the  above  modification  will  serve  to  detect  sugar  when  pre- 
sent in  as  small  quantity  as  0.015-0.02  per  cent.  This  claim  has  been 
corroborated  recently  by  Harrison.^  The  modified  solution  used  in  the 
above  test  differs  from  the  original  in  that  100  grams  of  sodium  car- 
bonate is  substituted  for  the  125  grams  of  potassium  hydroxide  ordi- 
narily used,  thus  forming  a  Fehling  solution  which  is  considerably  less 
alkaline  than  the  original.  This  alteration  in  the  composition  of  the 
Fehling  solution  is  of  advantage  in  the  detection  of  sugar  in  the  urine 
inasmuch  as  the  strong  alkalinity  of  the  ordinary  Fehling  solution  has  a 
tendency,  when  the  reagent  is  boiled  with  a  urine  containing  a  small 
amount  of  dextrose,  to  decompose  sufficient  of  the  sugar  to  render  the  de- 
tection of  the  remaining  portion  exceedingly  difficult  by  the  usual  technic. 
Benedict  claims  that  for  this  reason  the  use  of  his  modified  solution  per- 
mits the  detection  of  smaller  amounts  of  sugar  than  does  the  use  of  the 
ordinary  Fehling  solution.  Benedict  has  further  modified  his  solution 
for  use  in  the  quantitative  determination  of  sugar  (see  page  385). 

'  Benedict's  modified  Fehling  solution  consists  of  two  definite  solutions — a  copper  sulphate 
solution  and  an  alkaline  tartrate  solution,  which  may  be  prepared  as  follows: 

Copper  sulphate  solution  =$4.6$  grams  of  copper  suljjhate  dissolved  in  water  anrl  made  up 
to  500  c.c. 

Alkaline  tartrate  solution  =  ioo  grams  of  anhydrous  sodium  carbonate  and  173  grams  of 
Rochelle  salt  dissolved  in  water  anrl  made  uj)  to  500  c.c. 

These  sfjlutions  shoukl  be  preserved  separately  in  rubber-stoppered  bottles  and  mixed 
in  equal  volumes  when  needed  for  use.     This  is  done  to  prevent  deterioration. 

^  Harrison:  Pharm.  Jour.,  87,  746,  ic;ii. 


URINE.  329 

Second  Modification^ — \'cry  recently  Benedict  has  further  modi- 
fied his  solution  and  has  succeeded  in  obtaining  one  which  does  not 
deteriorate  upon  long  standing."  The  following  is  the  procedure  for 
the  detection  of  dextrose  in  the  urine:  To  5  c.c.  of  the  reagent  in  a 
test-tube  add  eight  (not  more)  drops  of  the  urine  to  be  examined.  The 
fluid  is  then  boiled  vigorously  for  from  one  to  two  minutes  and  then 
allowed  to  cool  sponlaneously.  In  the  presence  of  dextrose  the  entire 
body  of  the  solution  will  be  filled  with  a  precipitate,  which  may  be  red, 
yellow,  or  green  in  color,  depending  upon  the  amount  of  sugar  present. 
If  no  dextrose  is  present,  the  solution  will  either  remain  perfectly  clear, 
or  will  show  a  very  faint  turbidity,  due  to  precipitated  urates.  Even 
very  small  quantities  of  dextrose  in  urine  (o.i  per  cent)  yield  precipi- 
tates of  surprising  bulk  with  this  reagent,  and  the  positive  reaction  for 
dextrose  is  the  filling  of  the  entire  body  of  the  solution  with  a  precipitate, 
so  that  the  solution  becomes  opaque.  Since  amount  rather  than  color 
of  the  precipitate  is  made  the  basis  of  this  test,  it  may  be  applied,  even 
for  the  detection  of  small  quantities  of  dextrose,  as  readily  in  artificial 
light  as  in  daylight. 

{d)  Aliens  Modification  of  Fehling's  Test. — The  following  procedure 
is  recommended:  "From  7  to  8  c.c.  of  the  sample  of  urine  to  be  tested 
is  heated  to  boiling  in  a  test-tube,  and,  without  separating  any  precipi- 
tate of  albumin  which  may  be  produced,  5  c.c.  of  the  solution  of  cop- 
per sulphate  used  for  preparing  Fehling's  solution  is  added.  This  pro- 
duces a  precipitate  containing  uric  acid,  xanthine,  hypoxanthine,  phos- 
phates, etc.  To  render  the  precipitation  complete,  however,  it  is  desir- 
able to  add  to  the  liquid,  when  partially  cooled,  from  i  to  2  c.c.  of  a 
saturated  solution  of  sodium  acetate  having  a  feebly  acid  reaction  to 
litmus.'  The  liquid  is  filtered  and  to  the  filtrate,  which  will  have  a 
bluish-green  color,  5  c.c.  of  the  alkaline  tartrate  mixture  used  for  prepar- 
ing Fehling's  solution  is  added,  and  the  liquid  boiled  for  15-20  seconds. 

•  Benedict:  Jour.  .Am.  Med.  .Iss'n.,  57,  11Q3,  191 1. 

-  Benedict's  new  solution  has  the  following  composition: 

Copper  sulphate i7-3  S^- 

Sodium  citrate 173°  o^- 

Sodium  carbonate  (anhydrous) 100. o  gm. 

Distilled  water  to ." looo.o  c.c. 

With  the  aid  of  heat  dissolve  the  sodium  citrate  and  carbonate  in  about  600  c.c.  of  water. 
Pour  (through  a  folded  filter  if  necessary)  into  a  glass  graduate  and  make  up  to  850  c.c.  Dis- 
sohe  the  copper  sulphate  in  about  100  c.c  of  water  and  make  up  to  150  c.c.  Pour  the  carbonate- 
citrate  solution  into  a  large  beaker  or  casserole  and  add  the  copper  sulphate  solution  slowly, 
with  constant  stirring.  The  mixed  solution  is  ready  for  use,  and  does  not  deteriorate  upon 
long  standing. 

'  Sufficient  acetic  acid  should  be  added  to  the  sodium  acetate  solution  to  render  it  feebly 
acid  to  litmus.  A  saturated  solution  of  sodium  acetate  keeps  well,  but  weaker  solutions  are 
apt  to  become  mouldy,  and  then  possess  the  power  of  reducing  Fehling's  solution.  Hence 
it  is  essential  in  all  cases  of  importance  to  make  a  blank  test  by  mi.xing  equal  measures  of 
copper  sulphate  solution,  alkaline  tartrate  solution  and  water,  adding  a  little  sodium  acetate 
solution,  andhealing  the  mixture  to  boiling. 


330  PHYSIOLOGICAL    CHEMISTRY. 

In  the  presence  of  more  than  0.25  per  cent  of  sugar,  separation  of  cup- 
rous oxide  occurs  before  the  boiling-point  is  reached;  but  with  smaller 
quantities  precipitation  takes  place  during  the  cooling  of  the  solution, 
which  becomes  greenish,  opaque,  and  suddenly  deposits  cuprous  oxide 
as  a  fine  brownish-red  precipitate." 

(e)  Boettger^s  Test. — To  5  c.c.  of  urine  in  a  test-tube  add  i  c.c.  of 
KOH  or  NaOH  and  a  very  small  amount  of  bismuth  subnitrate,  and 
boil.  The  solution  will  gradually  darken  and  finally  assume  a  black 
color  due  to  reduced  bismuth.  If  the  test  is  made  with  urine  containing 
albumin  this  must  be  removed,  by  boiling  and  filtering,  before  applying 
the  test,  since  with  albumin  a  similar  change  of  color  is  produced  (bis- 
muth sulphide). 

(/)  Nylander^s  Test  {Almen's  Test). — To  5  c.c,  of  urine  in  a  test- 
tube  add  one-tenth  its  volume  of  Nylander's  reagent^  and  heat  for  five 
minutes  in  a  boiling  water-bath.^  The  mixture  will  darken  if  reducing 
sugar  is  present  and  upon  standing  for  a  few  moments  a  black  color 
will  appear.  This  color  is  due  to  the  precipitation  of  bismuth.  If  the 
test  is  made  on  urine  containing  albumin  this  must  be  removed,  by 
boiling  and  filtering,  before  applying  the  test,  since  with  albumin  a 
similar  change  of  color  is  produced.  Dextrose  when  present  to  the 
extent  of  0.08  per  cent  may  be  detected  by  this  reaction.  It  is  claimed 
by  Bechold  that  Nylander's  and  Boettger's  tests  give  a  negative  reaction 
with  solutions  containing  sugar  when  mercuric  chloride  or  chloroform 
is  present.  Other  observers^  have  failed  to  verify  the  inhibitory  action 
of  the  mercuric  chloride  and  have  shown  that  the  inhibitory  influence  of 
chloroform  may  be  overcome  by  raising  the  temperature  of  the  urine  to 
the  boiling-point  for  a  period  of  five  minutes  previous  to  making  the  test. 

Urines  rich  in  indican,  uroerythrin,  urochrome  or  hcsmato porphyrin, 
as  well  as  urines  excreted  after  the  ingestion  of  large  amounts  of  certain 
medicinal  substances,  may  give  a  darkening  of  Nylander's  reagent  similar 
to  that  of  a  true  sugar  reaction.  It  is  a  disputed  point  whether  the  urine 
after  the  administration  of  urotropin  will  reduce  Nylander's  reagent.'* 

Strausz  ^  has  recently  shown  that  the  urine  of  diabetics  to  whom 
"lothion"  (diiodohydroxypropane)  has  been  administered  will  give  a 
negative  Nylander's  reaction  and  respond  positively  to  the  Fehling  and 

'  Nylander's  reagent  is  prepared  by  digesting  2  grams  of  bismuth  subnitrate  and  4  grams 
of  Rochelle  salt  in  100  c.c.  of  a  10  per  cent  potassium  hydroxide  solution.  The  reagent  is 
then  cooled  and  filtered. 

^  Hammarsten  suggests  that  the  solution  be  boiled  for  2-5  minutes  (according  to  the  sugar 
content)  over  a  free  flame  and  the  tube  then  permitted  to  stand  five  minutes  before  drawing 
conclusions. 

^  Rehfuss  and  Hawk:  Jour.  Biol.  Chem.,  7,  267,  1910;  also  Zsidlitz:  Upsala  Lakdre- 
foren  Fork.,  N.  F.,  11,  1906. 

*  Abt:  Archives  of  Pediatrics,  24,  275,  1907;  also  Weitbrecht:  Schweiz.  Woch.,  47,  577,  1909. 

'Strausz:  Miinch  med.  Woch.,  59,  85,  1912. 


URINE.  331 

polarization  tests.  "lothion"  also  interferes  with  the  Xylander  test  in 
vitro  whereas  KI  and  I  do  not. 

According  to  Rustin  and  Otto  the  addition  of  PtCl^  increases  the 
delicacy  of  Xylander's  reaction.  They  claim  that  this  procedure  causes 
the  sugar  to  be  converted  quantitatively.  Xo  quantitative  method  has 
yet  been  devised,  however,  based  upon  this  principle. 

A  positive  Xylander  or  Boettger  test  is  probably  due  to  the  following 
reactions: 

{a)  Bi(OH)3(XO)3+KOH— Bi(OH)3+KX03. 

[h)  2Bi(OH)3-30— Bi,  +  3H20. 

Bohmansson,^  before  testing  the  urine  under  examination  treats  it 
(10  c.c.)  with  1/5  volume  of  25  per  cent  hydrochloric  acid  and  1/2 
volume  of  bone  black.  This  mixture  is  shaken  one  minute,  then  filtered, 
and  the  neutralized  filtrate  tested  by  X'ylander's  reaction.  Bohmansson 
claims  that  this  procedure  removes  certain  interfering  substances,  notably 
urochrome. 

0.  Fermentation  Test. — Rub  up  in  a  mortar  about  15  c.c.  of  the 
urine  with  a  small  piece  of  compressed  yeast.  Transfer  the  mixture  to  a 
saccharometer  (Fig.  3.  p.  36)  and  stand  it  aside  in  a  warm  place  for  about 
12  hours.  If  dextrose  is  present,  alcoholic  fermentation  will  occur  and 
carbon  dioxide  will  collect  as  a  gas  in  the  upper  portion  of  the  tube.  On 
the  completion  of  fermentation  introduce,  by  means  of  a  bent  pipette,  a 
little  KOH  solution  into  the  graduated  portion,  place  the  thumb  tightly 
over  the  opening  in  the  apparatus  and  invert  the  saccharometer.  Explain 
the  result. 

The  important  findings  of  X^euberg  and  associates^  recently  reported 
indicate  very  clearly  that  the  liberation  of  carbon  dioxide  by  yeast  is  not 
necessarily  a  criterion  of  the  presence  of  sugar.  The  presence  of  a  new 
enzyme  called  carboxylase  has  been  demonstrated  in  yeast  which  has  the 
power  of  splitting  off  CO^  from  the  carboxyl  graup  of  amino  and  other 
aliphatic  acids. 

7.  Barfoed's  Test. — Place  about  5  c.c.  of  Barfoed's  solution^  in 
a  test-tube  and  heat  to  boiling.  Add  the  urine  under  examination  slowly, 
a  lew  drops  at  a  time,  heating  after  each  addition.  Reduction  is  indi- 
cated by  the  production  of  a  red  precipitate.  If  the  precipitate  does  not 
form  upon  continued  boiling  allow  the  tube  to  stand  a  few  minutes  and 
examine.     XaCl  interferes  with  this  test  (Welker). 

Barfoed's  test  is  not  a  specific  test  for  dextrose  as  is  frequently  stated, 

'  Bohmansson:  Biochem.  Zeil.,  19,  p.  281. 

-  Neuberg  and  Associates:  Biochem.  Zeitsch.,  31,  170:  32,  323;  36,  (60,  68,  76),  1911. 
'  Barfoed's  solution  is  prepared  as  follows:     Dissolve  4.5  grams  of  neutral,  cr}-stallized 
copper  acetate  in  100  c.c.  of  water  and  add  1.2  c.c.  of  50  per  cent  acetic  acid. 


332  PHYSIOLOGICAL    CHEMISTRY. 

but  simply  serves  to  detect  monosaccharides.  Disaccharides  will  also 
respond  to  the  test,  according  to  Hinkel  and  Sherman/  if  the  solutionis 
boiled  sufficiently  long  in  contact  with  the  reagent  to  hydrolyze  the  disac- 
charide  through  the  action  of  the  acetic  acid  present  in  the  Barfoed's 
solution. 

Mathews  and  McGuigan  ^  have  also  shown  that  disaccharides  will 
respond  to  this  test  under  proper  conditions  of  acidity. 

8.  Polariscopic  Examination. — For  directions  as  to  the  use  of  the 
polariscope  see  page  36. 

PROTEINS. 

Normal  urine  contains  a  trace  of  protein  material  but  the  amount 
present  is  so  slight  as  to  escape  detection  by  any  of  the  simple  tests  in 
general  use  for  the  detection  of  protein  urinary  constituents.  The 
following  are  the  more  important  forms  of  protein  material  which  have 
been  detected  in  the  urine  under  pathological  conditions: 

(i)  Serum  albumin. 

(2)  Serum  globulin. 

r  Deutero-proteose. 

(3)  Proteoses    |   Hetero-proteose. 

I  "Bence- Jones'  protein." 

(4)  Peptone. 

(5)  Nucleoprotein. 

(6)  Fibrin. 

(7)  Oxyhaemoglobin. 

ALBUMIN. 

Albuminuria  is  a  condition  in  which  serum  albumin  or  serum  globulin 
appears  in  the  urine.  There  are  two  distinct  forms  of  albuminuria,  i.  e., 
renal  albuminuria  and  accidental  albuminuria.  Sometimes  the  terms 
"true"  albuminuria  and  "false"  albuminuria  are  substituted  for  those 
just  given.  In  the  renal  type  the  albumin  is  excreted  by  the  kidneys. 
This  is  the  more  serious  form  of  the  malady  and  at  the  same  time  is  more 
frequently  encountered  than  the  accidental  type.  Among  the  causes  of 
renal  albuminuria  are  altered  blood  pressure  in  the  kidneys,  altered 
kidney  structure,  or  changes  in  the  composition  of  the  blood  entering 
the  kidneys,  thus  allowing  the  albumin  to  diffuse  more  readily.  In  the 
accidental  form  of  albuminuria  the  albumin  is  not  excreted  by  the  kidneys 
as  is  the  case  in  the  renal  form  of  the  disorder,  but  arises  from  the  blood, 

'  Hinkle  &  Sherman:     Journ.  Am.  Chem.  Soc,  29,  1744,  1907. 
'^  Mathews  and  McGuigan:     A mer.  Journ.  Physiol.,  19,  175,  1907. 


URINE,  ^;^s 

lymph,  or  some  al])umin-containmg  exudate  coming  into  contact  with  the 
urine  at  some  point  below  the  kidneys. 

Experiments. 

Heller's  Ring  Test. — Place  5  c.c.  of  concentrated  HNO3  ^^  ^  test- 
tube,  incline  the  tube,  and,  by  means  of  a  pipette  allow  the  urine  to  flow 
slowly  down  the  side.  The  liquids  should  stratify  with  the  formation  of 
a  while  zone  of  precipitated  albumin  at  the  point  of  juncture.  If  the 
albumin  is  present  in  very  small  amount  the  white  zone  may  not  form  until 
the  tube  has  been  allowed  to  stand  for  several  minutes.  If  the  urine  is 
quite  concentrated  a  white  zone,  due  to  uric  acid  or  urates,  will  form 
upon  treatment  with  nitric  acid  as  indicated.  This  ring  may  be  easily 
differentiated  from  the  albumin  ring  by  repeating  the  test  after  diluting 
the  urine  with  3  or  4  volumes  of  water,  whereupon  the  ring,  if  due  to  uric 
acid  or  urates,  will  not  appear.  It  is  ordinarily  possible  to  differentiate 
between  the  albumin  ring  and  the  uric  acid  ring  without  diluting  the 
urine,  since  the  ring,  when  due  to  uric  acid,  has  ordinarily  a  less  sharply 
defined  upper  border,  is  generally  broader  than  the  albumin  ring  and  fre- 
quently is  situated  in  the  urine  above  the  point  of  contact  with  the  nitric 
acid.  Concentrated  urines  also  occasionally  exhibit  the  formation,  at 
the  point  of  contact,  of  a  crystalline  ring  with  very  sharply  defined  borders. 
This  is  urea  nitrate  and  is  easily  distinguished  from  the  "fluffy"  ring  of 
albumin.  If  there  is  any  difficulty  in  differentiation  a  simple  dilution  of 
the  urine  with  water,  as  above  described,  will  remove  the  difficulty. 
Various  colored  zones,  due  cither  to  the  presence  of  indican,  bile  pigments, 
or  to  the  oxidation  of  other  organic  urinary  constituents,  may  form  in 
this  test  under  certain  conditions.  These  colored  rings  should  never 
be  confounded  with  the  white  ring  which  alone  denotes  the  presence  of 
albumin. 

After  the  administration  of  certain  drugs  a  white  precipitate  of  resin 
acids  may  form  at  the  point  of  contact  of  the  two  fluids  and  may  cause 
the  observer  to  draw  wrong  conclusions.  This  ring,  if  composed  of 
resin  acids,  will  dissolve  in  alcohol,  whereas  the  albumin  ring  will  not 
dissolve. 

Weinberger  has  recently  shown  that  a  ring  closely  resembling  the 
albumin  ring  is  often  obtained  in  urines  preserved  by  thymol  when  sub- 
jected to  Heller's  test.  The  ring  is  due  to  the  formation  of  nitrosothymol 
and  possibly  nitrothymol.  If  the  thymol  is  removed  from  the  urine  by 
extraction  with  petroleum  ether^  previous  to  adding  nitric  acid,  the  ring 
does  not  form. 

*  Accomplished  readily  by  gently  agitating  equal  volumes  of  petroleum  ether  and  the 
urine  under  examination  for  two  minutes  in  a  test-tube  before  applying  the  test. 


334  PHYSIOLOGICAL   CHEMISTRY. 

An  instrument  called  the  alhumoscope  Qiorismascope)  has  been  devised 
for  use  in  this  test  and  has  met  with  considerable  favor.  The  method  of 
using  the  albumoscope  is  described  below. 

Use  oj  the  Alhumoscope. — This  instrument  is  intended  to  facilitate 
the  making  of  "ring"  tests  such  as  Heller's  and  Roberts'.  In  making 
a  test  about  5  c.c.  of  the  solution  under  examination  is  first  introduced 
into  the  apparatus  through  the  larger  arm  and  the  reagent  used  in  the 
particular  test  is  then  introduced  through  the  capillary  arm  and  allowed 
to  flow  down  underneath  the  solution  under  examination.  If  a  reason- 
able amount  of  care  is  taken  there  is  no  possibility  of  mixing  the  two 
solutions  and  a  definitely  defined  white  "ring"  is  easily  obtained  at  the 
zone  of  contact. 

2.  Roberts'  Ring  Test. — ^Place  5  c.c.  of  Roberts'  reagent^  in  a  test- 
tube,  incline  the  tube,  and,  by  means  of  a  pipette  allow  the  urine  to  flow 
slowly  down  the  side.  The  liquids  should  stratify  with  the  formation  of  a 
while  zone  of  precipitated  albumin  at  the  point  of  juncture.  This  test  is 
a  modification  of  Heller's  ring  test  and  is  rather  more  satisfactory  than 
that  test,  since  the  colored  rings  never  form  and  the  consequent  confusion 
is  avoided.  The  alhumoscope  (see  above)  may  also  be  used  in  making 
this  test. 

3.  Spiegler's  Ring  Test. — Place  5  c.c.  of  Spiegler's  reagent^  in  a  test- 
tube,  incline  the  tube,  and,  by  means  of  a  pipette,  allow  5  c.c.  of  urine, 
acidified  with  acetic  acid,  to  flow  slowly  down  the  side.  A  white  zone 
will  form  at  the  point  of  contact.  This  is  an  exceedingly  delicate  test,  in 
fact  too  delicate  for  ordinary  clinical  purposes,  since  it  serves  to  detect 
albumin  when  present  in  the  merest  trace  (1:250,000)  and  hence  most 
normal  urines  will  give  a  positive  reaction  for  albumin  when  this  test  is 
applied. 

Some  investigators  claim  that  the  delicacy  of  this  test  depends  upon  the 
presence  of  sodium  chloride  in  the  urine,  the  test  losing  accuracy  if  the 
sodium  chloride  content  be  low. 

4.  Jolles'  Reaction. — Shake  5  c.c.  of  urine  with  i  c.c.  of  30  per  cent 
acetic  acid  and  4  c.c.  of  Jolles'  reagent^  in  a  test-tube.  A  white  precipitate 
indicates  the  presence  of  albumin. 

•  Roberts'  reagent  is  composed  of  i  volume  of  concentrated  HNO3  and  5  volumes  of  a 
saturated  solution  of  MgSO^. 

^  Spiegler's  reagent  has  the  following  composition: 

Tartaric  acid 20  grams. 

Mercuric  chloride 40  grams. 

Glycerol 100  grams. 

Distilled  water 1000  grams. 

*  Jolles'  reagent  has  the  following  composition: 

Succinic  acid 4°  grams. 

Mercuric  chloride 20  grams. 

Sodium  chloride 20  grams. 

Distilled  water 1000  grams. 


URINE.  335 

Care  should  be  taken  to  use  the  correct  amount  of  acetic  acid,  since  the 
use  of  too  small  an  amount  may  result  in  the  formation  of  mercury  com- 
binations which  may  cause  confusion.  In  the  presence  of  iodine,  mer- 
curic iodide  will  form  but  may  readily  be  diflerentiated  from  albumin 
through  the  fact  that  it  is  soluble  in  alcohol. 

5.  Coagulation  or  Boiling  Test. — (a)  Heat  5  c.c.  of  urine  to 
boiling  in  a  test-tube.  A  precipitate  forming  at  this  point  is  due  either 
to  albumin  or  to  phosphates.  Acidify  the  urine  slightly  by  the  addition 
of  3-5  drops  of  very  dilute  acetic  acid,  adding  the  acid  drop  by  drop 
to  the  hot  solution.  If  the  precipitate  is  due  to  phosphates  it  will  dis- 
appear under  these  conditions,  whereas  if  it  is  due  to  albumin  it  will 
not  only  fail  to  disappear  but  will  become  more  fiocculent  in  character, 
since  the  reaction  of  a  fluid  must  be  acid  to  secure  the  complete  pre- 
cipitation of  the  albumin  by  this  coagulation  process.  Too  much  acid 
should  be  avoided  since  it  will  cause  the  albumin  to  go  into  solution. 
Certain  resin  acids  may  be  precipitated  by  the  acid,  but  the  precipitate 
due  to  this  cause  may  be  easily  differentiated  from  the  albumin  pre- 
cipitate by  reason  of  its  solubility  in  alcohol. 

{h)  A  modification  of  this  test  in  quite  general  use  is  as  follows: 
Fill  a  test-tube  two-thirds  full  of  urine  and  gently  heat  the  upper  half 
of  the  fluid  to  boiling,  being  careful  that  this  fluid  does  not  mix  with 
the  lower  half.  A  turbidity  indicates  albumin  or  phosphates.  Acidify 
the  urine  slightly  by  the  addition  of  3-5  drops  of  dilute  acetic  acid,  when 
the  turbidity,  if  due  to  phosphates,  will  disappear. 

Nitric  acid  is  often  used  in  place  of  acetic  acid  in  these  tests.  In 
case  nitric  acid  is  used  ordinarily  1-2  drops  is  sufficient. 

6.  Acetic  Acid  and  Potassium  Ferrocyanide  Test. — To  5  c.c. 
of  urine  in  a  test-tube  add  5-10  drops  of  acetic  acid.  Mix  well  and 
add  potassium  ferrocyanide  drop  by  drop,  until  a  preciptate  forms. 
This  is  a  very  delicate  test.  Schmiedl  claims  that  a  precipitate  of 
Fe(Cn)gK2Zn  or  Fe(Cn)8Zn2  is  formed  when  urines  containing  zinc 
are  subjected  to  this  test  and  that  this  precipitate  resembles  the  pre- 
cipitate secured  with  protein  solutions.  In  the  case  of  human  urine  a 
reaction  was  obtained  when  0.000022  gram  of  zinc  per  cubic  centimeter 
was  present.  Schmiedl  further  found  that  the  urine  collected  from 
rabbits  housed  in  zinc-lined  cages  possessed  a  zinc  content  which  was 
sufficient  to  yield  a  ready  response  to  the  test.  Zinc  is  the  only  interfering 
substance  so  far  reported. 

7.  Tanret's  Test. — To  5  c.c.  of  urine  in  a  test-tube  add  Tarnet's 
reagent^   drop   by   drop  until   a  turbidity  or  precipitate  forms.     This 

*  Tanret's  reagent  is  prepared  as  follows:  Dissolve  1.35  gram  of  mercuric  chloride  in 
25  c.c.  of  water,  add  to  this  solution  3.32  grams  of  potassium  iodide  dissolved  in  25  c.c.  of 
water,  then  make  the  total  soludon  up  to  60  c.c.  with  water  and  add  20  c.c.  of  glacial  acetic 
acid  to  the  mixture. 


336  PHYSIOLOGICAL   CHEMISTRY. 

is  an  exceedingly  delicate  test.  Sometimes  the  urine  is  stratified  upon 
the  reagent  as  in  Heller's  or  Roberts'  ring  test.  According  to  Repiton, 
urates  interfere  with  the  delicacy  of  this  test.  Tanret,  however,  claims 
that  urates  do  not  interfere  inasmuch  as  any  precipitate  due  to  urates 
may  be  brought  into  solution  by  heat  whereas  an  albumin  precipitate 
under  the  same  conditions  will  persist.  Tanret  further  states  that 
mucin  interferes  with  the  delicacy  of  the  test  and  that  it  should  therefore 
be  removed  from  the  urine  under  examination  by  acidification  with 
acetic  acid  and  filtration  before  testing  for  albumin. 

8.  Sodium  Chloride  and  Acetic  Acid  Test. — Mix  two  volumes 
of  urine  and  one  volume  of  a  saturated  solution  of  sodium  chloride 
in  a  test-tube,  acidify  with  acetic  acid,  and  heat  to  boiling.  The  pro- 
duction of  a  cloudiness  or  the  formation  of  a  precipitate  indicates  the 
presence  of  albumin.  The  resin  acids  may  interfere  here  as  in  the 
ordinary  coagulation  test  (page  335),  but  they  may  be  easily  differentiated 
from  albumin  by  means  of  their  solubility  in  alcohol. 

9.  Potassium  Iodide  Test/ — Dilute  5  c.c.  of  the  urine  under 
examination  with  10  c.c.  of  water  and  stratify  this  mixture  upon  a 
potassium  iodide  solution  made  slightly  acid  with  acetic  acid.  In 
the  presence  of  0.01-0.02  per  cent  of  albumin  a  white  ring  forms  imme- 
diately. If  the  test  be  allowed  to  stand  two  minutes  after  the  stratifi- 
cation it  will  serve  to  detect  0.005  per  cent  of  albumin. 

GLOBULIN. 

Serum  globulin  is  not  a  constituent  of  normal  urine  but  frequently 
occurs  in  the  urine  under  pathological  conditions  and  is  ordinarily 
associated  with  serum  albumin.  In  albuminuria  globulin  in  varying 
amounts  often  accompanies  the  albumin,  and  the  clinical  significance 
of  the  two  is  very  similar.  Under  certain  conditions  globulin  may  occur 
in  the  urine  unaccompanied  by  albumin. 

Experiments. 

Globulin  will  respond  to  all  the  tests  just  outlined  under  Albumin. 
If  it  is  desirable  to  differentiate  between  albumin  and  globulin  in  any 
urine  the  following  processes  may  be  employed: 

I.  Saturation  with  Magnesium  Sulphate.— Place  25  c.c.  of  neutral 
urine  in  a  small  beaker  and  add  pulverized  magnesium  sulphate  in 
substance  to  the  point  of  saturation.  If  the  protein  present  is  globulin 
it  will  precipitate  at  this  point.  If  no  precipitate  is  produced  acidify 
the  saturated  solution  with  acetic  acid  and  warm  gently.  Albumin  will 
be  precipitated  if  present. 

The  above  procedure  may  be  used  to  separate  globulin  and  albumin 

'  Pharm.  Ztg.,  54,  p.  612. 


URINE.  337 

if  present  in  the  same  urine.  To  do  this  filter  off  the  globulin  after  it 
has  been  precipitated  by  the  magnesium  sulphate,  then  acidify  the  clear 
solution  and  warm  gently  as  directed.  Note  the  formation  of  the  albumin 
precipitate. 

2.  Half-saturation  with  Ammonium  Sulphate. — Place  25  c.c. 
of  neutral  urine  in  a  small  beaker  and  add  an  equal  volume  of  a  satu- 
rated solution  ^of  ammonium  sulphate.  Globulin,  if  present,  will  be 
precipitated.  If  no  precipitate  forms  add  ammonium  sulphate  m  sub- 
stance to  the  point  of  saturation.  If  albumin  is  present  it  will  be  pre- 
cipitated upon  saturation  of  the  solution  as  just  indicated.  This 
method  may  also  be  used  to  separate  globulin  and  albumin  when  they 
occur  in  the  same  urine. 

Frequently  in  urine  which  contains  a  large  amount  of  urates  a  pre- 
cipitate of  ammonium  urate  may  occur  when  the  ammonium  sulphate 
solution  is  added  to  the  urine.  This  urate  precipitate  should  not  be 
confounded  with  the  precipitate  due  to  globulin.  The  two  precipitates 
may  be  differentiated  by  means  of  the  fact  that  the  urate  precipitate 
ordinarily  appears  only  after  the  lapse  of  several  minutes  whereas  the 
globulin  generally  precipitates  at  once. 

PROTEOSE  AND  PEPTONE. 

Proteoses,  particularly  deutero-proteose  and  hetero-proteose,  have 
frequently  been  found  in  the  urine  under  various  pathological  con- 
ditions such  as  diphtheria,  pneumonia,  intestinal  ulcer,  carcinoma, 
dermatitis,  osteomalacia,  atrophy  of  the  kidneys,  and  in  sarcomata 
of  the  bones  of  the  trunk.  "Bence-Jones'  protein,"  a  proteose-like 
substance,  is  of  interest  in  this  connection  and  its  appearance  in  the 
urine  is  believed  to  be  of  great  diagnostic  importance  in  cases  of  multi- 
ple myeloma  or  myelogenic  osteosarcoma.  By  some  investigators  this 
protein  is  held  to  be  a  variety  of  hetero-proteose  whereas  others  claim 
that  it  possesses  albumin  characteristics. 

Peptone  certainly  occurs  much  less  frequently  as  a  constituent  of 
the  urine  than  does  proteose,  in  fact  most  investigators  seriously  ques- 
tion its  presence  under  any  conditions.  There  are  many  instances 
of  peptonuria  cited  in  the  early  literature,  but  because  of  the  uncertainty 
in  the  conception  of  what  really  constituted  a  peptone  it  is  probable  that 
in  many  cases  of  so-called  peptonuria  the  protein  present  was  really 
proteose. 

Experiments. 

I.  Boiling  Test. — Make  the  ordinary  coagulation  test  according 
to  the  directions  given  under  Albumin,  page  335.     If  no  coagulable 


338  PHYSIOLOGICAL   CHEMISTRY. 

protein  is  found  allow  the  boiled  urine  to  stand  and  note  the  gradual 
appearance,  in  the  cooled  fluid,  of  a  flaky  precipitate  of  proteose.  This 
is  a  crude  test  and  should  never  be  relied  upon. 

2.  Schulte's  Method. — Acidify  50  c.c.  of  urine  with  dilute  acetic 
acid  and  filter  off  any  precipitate  of  nucleoprotein  which  may  form. 
Now  test  a  few  cubic  centimeters  of  the  urine  for  coagulable  protein,  by 
tests  2  and  5  under  Albumin,  pp.  334-5.  If  coagulable  protein, is  present 
remove  it  by  coagulation  and  filtration  before  proceeding.  Introduce 
25  c.c.  of  the  urine,  freed  from  coagulable  protein,  into  150  c.c.  of  absolute 
alcohol  and  allow  it  to  stand  for  12-24  hours.  Decant  the  supernatant 
fluid  and  dissolve  the  precipitate  in  a  small  amount  of  hot  water.  Now 
filter  this  solution,  and  after  testing  again  for  nucleoprotein  with  very 
dilute  acetic  acid,  try  the  biuret  test.  If  this  test  is  positive  the  presence 
of  proteose  is  indicated.^ 

Urobilin  does  not  ordinarily  interfere  with  this  test  since  it  is  almost 
entirely  dissolved  by  the  absolute  alcohol  when  the  proteose  is  precipitated. 

3.  V.  Alder's  Method. — Acidify  10  c.c.  of  urine  with  hydrochloric 
acid,  add  phosphotungstic  acid  until  no  more  precipitate  forms  and 
centrifugate^  the  solution.  Decant  the  supernatant  fluid,  add  some 
absolute  alcohol  to  the  precipitate,  and  centrifugate  again.  This  washing 
with  alcohol  is  intended  to  remove  the  urobilin  and  hence  should  be  con- 
tinued so  long  as  the  alcohol  exhibits  any  coloration  whatever.  Now 
suspend  the  precipitate  in  water  and  add  potassium  hydroxide  to  bring  it 
into  solution.  At  this  point  the  solution  may  be  blue  in  color,  in  which 
case  decolorization  may  be  secured  by  gently  heating.  Apply  the  biuret 
test  to  the  cool  solution.  A  positive  biuret  test  indicates  the  presence  of 
proteoses. 

4.  Detection  of  '' Bence-Jones'  Protein." — Heat  the  suspected 
urine  very  gently,  carefully  noting  the  temperature.  At  as  low  a  tem- 
perature as  40°  C.  a  turbidity  may  be  observed,  and  as  the  temperature  is 
raised  to  about  60°  C.  a  flocculent  precipitate  forms  and  clings  to  the  sides 
of  the  test-tube.  If  the  urine  is  now  acidified  very  slightly  with  acetic 
acid  and  the  temperature  further  raised  to  100°  C.  the  precipitate  at  least 
partly  disappears;  it  will  return  upon  cooling  the  tube. 

This  property  of  precipitating  at  so  low  a  temperature  and  of  dis- 
solving at  a  higher  temperature  is  typical  of  "Bence- Jones'  protein"  and 
may  be  used  to  differentiate  it  from  all  other  forms  of  protein  material 
occurring  in  the  urine. 

^  If  it  is  considered  desirable  to  test  for  peptone  the  proteose  may  be  removed  by  satu- 
ration with  (NHJ2SO4  according  to  the  directions  given  on  page  120  and  the  filtrate  tested 
for  peptone  by  the  biuret  test. 

^  It  not  convenient  to  use  a  centrifuge  the  precipitate  may  be  filtered  off  and  washed  on 
the  fiUer  paper  with  alcohol. 


URINE.  339 

NUCLEOPROTEIN. 

There  has  been  considerable  controversy  as  to  the  proper  classification 
for  the  protein  body  which  forms  the  "nubecula"  of  normal  urine.  By 
different  investigators  it  has  been  called  mucin,  mucoid,  phospho protein, 
nuclcoaUnimin,  and  nuclcoprotein.  Of  course,  according  to  the  modern 
acceptation  of  the  meanings  of  these  terms  they  cannot  be  synonymous. 
Mucin  and  mucoid  arc  glycoproteins  and  hence  contain  no  phosphorus 
(see  p.  1 12),  whereas  phosphoproteins  and  nucleoproteins  are  phos- 
phorized  bodies.  It  may  possibly  be  that  both  these  forms  of  protein, 
i.  e.,  the  glycoprotein  and  the  phosphorized  type,  ocCur  in  the  urine  under 
certain  conditions  (see  page  308).  In  this  connection  we  will  use  the 
term  nuclcoprotein.  The  pathological  conditions  under  which  the  content 
of  nuclcoprotein  is  increased  includes  all  affections  of  the  urinary  passages 
and  in  particular  pyelitis,  nephritis,  and  inflammation  of  the  bladder. 

Experiments. 

1.  Detection  of  Nucleoprotein. — Place  10  c.c.  of  urine  in  a  small 
beaker,  dilute  it  with  three  volumes  of  water  to  prevent  precipitation  of 
urates,  and  make  the  reaction  very  strongly  acid  with  acetic  acid.  If  the 
urine  becomes  turbid  it  is  an  indication  that  nucleoprotein  is  present. 

If  the  urine  under  examination  contains  albumin  the  greater  portion 
of  this  substance  should  be  removed  by  boiling  the  urine  before  testing  it 
for -the  presence  of  nucleoprotein. 

2.  Ott's  Precipitation  Test. — Mix  25  c.c.  of  the  urine  with  an  equal 
volume  of  a  saturated  solution  of  sodium  chloride  and  slowly  add  Almen's 
reagent.^  In  the  presence  of  nucleoprotein  a  voluminous  precipitate 
forms. 

BLOOD. 

The  pathological  conditions  in  which  blood  occurs  in  the  urine  m^y  be 
classified  under  the  two  divisions  hcpmaturia  and  hcemoglohinuria.  In 
haematuria  we  are  able  to  detect  not  only  the  haemoglobin  but  the  unrup- 
tured corpuscles  as  well,  whereas  in  haemoglobinuria  the  pigment  alone  is 
present.  Haematuria  is  brought  about  through  blood  passing  into  the 
urine  because  of  some  lesion  of  the  kidney  or  of  the  urinary  tract  below 
the  kidney.  Haemoglobinuria  is  brought  about  through  haemolysis,  i.  e., 
the  rupturing  of  the  stroma  of  the  erythrocyte  and  the  liberation  of  the 
haemoglobin.  This  may  occur  in  scur\y,  typhus,  pyemia,  purpura,  and 
in  other  diseases.     It  may  also  occur  as  the  result  of  a  burn  covering  a 

'  Dissolve  5  grams  of  tannin  in  240  c.c.  of  50  per  cent  alcohol  and  add  10  c.c.  of  25  per 
cent  acetic  acid. 


340  PHYSIOLOGICAL   CHEMISTRY, 

considerable  area  of  the  body,  or  may  be  brought  about  through  the 
action  of  certain  poisons  or  by  the  injections  of  various  substances  having 
the  power  of  dissolving  the  erythrocytes.  Transfusion  of  blood  may  also 
cause  hasmoglobinuria. 

Experiments. 

1.  Heller's  Test. — Render  lo  c.c.  of  urine  strongly  alkaline  with 
potassium  hydroxide  solution  and  heat  to  boiling.  Upon  allowing  the 
heated  urine  to  stand  a  precipitate  of  phosphates,  colored  red  by  the 
contained  haematin,  is  formed.  It  is  ordinarily  well  to  make  a  "control" 
experiment  using  normal  urine,  before  coming  to  a  final  decision. 

Certain  substances,  such  as  cascara  sagrada,  rhubarb,  santonin,  and 
senna,  cause  the  urine  to  give  a  similar  reaction.  Reactions  due  to  such 
substances  may  be  differentiated  from  the  true  blood  reaction  by  the 
fact  that  both  the  precipitate  and  the  pigment  of  the  former  reaction 
disappear  when  treated  with  acetic  acid,  whereas  if  the  color  is  due  to 
haematin  the  acid  will  only  dissolve  the  precipitate  of  phosphates  and 
leave  the  pigment  undissolved. 

2.  Teichmann's  Haemin  Test. — Place  a  small  drop  of  the  suspected 
urine  or  a  small  amount  of  the  moist  sediment  on  a  microscopic  slide, 
add  a  minute  grain  of  sodium  chloride  and  carefully  evaporate  to  dryness 
over  a  low  flame.  Put  a  cover  glass  in  place,  run  underneath  it  a  drop  of 
glacial  acetic  acid,  and  warm  gently  until  the  formation  of  gas  bubbles 
is  observed.  Cool  the  preparation,  examine  under  the  microscope,  and 
compare  the  form  of  .the  crystals  with  those  reproduced  in  Figs.  59  and 
60,  page  211.     (See  Atkinson  and  Kendall's  modification,  p.   210.) 

3.  Heller-Teichmann  Reaction.^ — Produce  the  pigmented  pre- 
cipitate according  to  directions  given  in  Heller's  test  above.  If  there  is 
a  copious  precipitate  of  phosphates  and  but  little  pigment  the  phosphates 
may  be  dissolved  by  treatment  with  acetic  acid  and  the  residue  used  in  the 
formation  of  the  haemin  crystals  according  to  directions  in  Experiment  2, 
above. 

4.  V.  Zeynek  and  Nencki's  Haemin  Test. — To  10  c.c.  of  the  urine 
under  examination  add  acetone  until  no  more  precipitate  forms.  Filter 
off  the  precipitate  and  extract  it  with  10  c.c.  of  acetone  rendered  acid 
with  2-3  drops  of  hydrochloric  acid.  Place  a  drop  of  the  resulting  colored 
extract  on  a  slide,  immediately  place  a  cover  glass  in  position,  and  examine 
under  the  microscope.  Compare  the  form  of  the  crystals  with  those 
shown  in  Figs.  59  and  60,  page  211.  Haemin  crystals  produced  by  this 
manipulation  are  sometimes  very  minute,  thus  rendering  it  difficult  to 
determine  the  exact  form  of  the  crystal. 

5.  Schalfijew's  Haemin  Test. — Place  20  c.c.  of  glacial  acetic  acid  in 


URINE.  341 

a  small  beaker  and  heat  to  80°  C.  Add  5  c.c.  of  the  urine  under  examina- 
tion, raise  the  temperature  to  80°  C,  and  stand  the  mixture  aside  to  cool. 
Examine  the  crystals  under  the  microscope  and  compare  them  with  those 
shown  in  Figs.  59  and  60,  page  211. 

6.  Guaiac  Test. — Place  5  c.c.  of  urine  in  a  test-tube  and  by  means 
of  a  pipette  introduce  a  freshly  prepared  alcoholic  solution  of  guaiac 
(strength  about  1:60)  into  the  fluid  until  a  turbidity  results,  then  add 
old  turpentine  or  hydrogen  peroxide,  drop  by  drop,  until  a  blue  color  is 
obtained.  This  is  a  very  delicate  test  when  properly  performed.  Buck- 
master  has  recently  suggested  the  use  of  guaiaconic  acid  instead  of  the 
solution  of  guaiac.     See  discussion  on  page  204  and  test  on  page  209. 

7.  Schumm's  Modification  of  the  Guaiac  Test. — To  about  5  c.c. 
of  urine'  in  a  test-tube  add  about  10  drops  of  a  freshly  prepared  alcoholic 
solution  of  guaiac.  Agitate  the  tube  gently,  add  about  20  drops  of  old 
turpentine,  subject  the  tube  to  a  thorough  shaking,  and  permit  it  to  stand 
for  about  2-3  minutes.  A  blue  color  indicates  the  presence  of  blood  in 
the  solution  under  examination.  In  case  there  is  not  sufficient  blood  to 
yield  a  blue  color  under  these  conditions,  a  few  c.c.  of  alcohol  should  be 
added  and  the  tube  gently  shaken,  whereupon  a  blue  coloration  will 
appear  in  the  upper  alcohol-turpentine  layer. 

A  control  test  should  always  be  made  using  water  in  place  of  urine. 
In  the  detection  of  very  minute  traces  of  blood  only  3-5  drops  of  the 
guaiac  solution  should  be  employed. 

8.  Adler's  Benzidine  Reaction. — This  is  one  of  the  most  delicate  of 
the  reactions  for  the  detection  of  blood.  Different  benzidine  preparations 
vary  greatly  in  their  sensitiveness,  however.  Inasmuch  as  benzidine 
solutions  change  readily  upon  contact  with  light,  it  is  essential  that  they 
be  kept  in  a  dark  place.  The  test  is  performed  as  follows :  To  a  saturated 
solution  of  benzidine  in  alcohol  or  glacial  acetic  acid  add  an  equal  volume 
of  3  per  cent  hydrogen  peroxide  and  i  c.c.  of  the  urine  under  examination. 
If  the  mixture  is  not  already  acid,  render  it  so  with  acetic  acid,  and  note 
the  appearance  of  a  green  or  blue  color.  A  control  test  should  be  made 
substituting  water  for  the  urine. 

Often  when  urines  containing  a  small  amount  of  blood  are  tested  by 
this  reaction,  the  mixture  is  rendered  so  turbid  as  to  make  it  diflScult  to 
decide  as  to  the  presence  of  a  faint  green  color.  Such  urines  should  be 
extracted  with  an  ether-acetic  acid  solution  and  the  resulting  extract 
washed  with  water  before  the  test  is  applied  to  it.  The  sensitiveness  of 
the  benzidine  reaction  is  greater  when  applied  to  aqueous  solutions  than 
when  applied  to  the  urine. 

'  Alkaline  urine  should  be  made  slighdy  acid  with  acetic  acid  as  the  blue  end-reaction 
is  very  sensitive  to  alkali. 


342  PHYSIOLOGICAL    CHEMISTRY. 

9.  Spectroscopic  Examination. — Submit  the  urine  to  a  spectro- 
scopic examination  according  to  the  directions  given  on  page  215,  looking 
especially  for  the  absorption-bands  of  oxyhemoglobin  andmethsemoglobin 
(see  Absorption  Spectra,  Plate  I.). 

BILE. 

Both  the  pigments  and  the  acids  of  the  bile  may  be  detected  in  the 
urine  under  certain  pathological  conditions.  Of  the  pigments,  bilirubin 
is  the  only  one  which  has  been  positively  identified  in  fresh  urine;  the 
other  pigments,  when  present,  are  probably  derived  from  the  bilirubin. 
A  urine  containing  bile  may  be  yellowish-green  to  brown  in  color  and 
when  shaken  foams  readily.  The  staining  of  the  various  tissues  of  the 
body  through  the  absorption  of  bile  due  to  occlusion  of  the  bile  duct 
cause  a  condition  known  as  icterus  or  jaundice.  Bile  is  always  present  in 
the  urine  under  such  conditions  unless  the  amount  of  bile  reaching  the 
tissues  is  extremely  small. 

Experiments. 

Tests  for  Bile  Pigments. 

1.  Gmelin's  Test. — To  about  5  c.c.  of  concentrated  nitric  acid  in  a 
test-tube  add  an  equal  volume  of  urine  carefully  so  that  the  two  fluids  do 
not  mix.  At  the  point  of  contact  note  the  various  colored  rings,  green,  blue, 
violet,  red,  and  reddish-yellow. 

2.  Rosenbach's  Modification  of  Gmelin's  Test.^ — Filter  5  c.c.  of 
urine  through  a  small  filter  paper.  Introduce  a  drop  of  concentrated  nitric 
acid  into  the  cone  of  the  paper  and  observe  the  succession  of  colors  as 
given  in  Gmelin's  test. 

3.  Nakayama's  Reaction. — To  5  c.c.  of  urine  in  a  test-tube  add  an 
equal  volume  of  a  10  per  cent  solution  of  barium  chloride.  Centrifugate 
the  mixture,  pour  off  the  supernatant  fluid,  and  heat  the  precipitate  with 
2  c.c.  of  Nakayama's  reagent.^  In  the  presence  of  bile  pigments  the 
solution  assumes  a  blue  or  green  color. 

3.  Huppert's  Reaction. — Thoroughly  shake  equal  volumes  of  urine 
and  milk  of  lime  in  a  test-tube.  The  pigments  unite  with  the  calcium 
and  are  precipitated.  Filter  off  the  precipitate,  wash  it  with  water,  and 
transfer  to  a  small  beaker.  Add  alcohol  acidified  slightly  with  hydro- 
chloric acid  and  warm  upon  a  water-bath  until  the  solution  becomes 
colored  an  emerald  green. 

According  to   Steensma,  this  procedure  may  give  negative  results 

'  Prepared  by  combining  99  c.c.  of  alcohol  and  i  c.c.  of  fuming  hydrochloric  acid  con- 
taining 4  grams  of  ferric  chloride  per  liter. 


URINE.  343 

even  in  the  presence  of  the  pigments,  owing  to  the  fact  that  the  acid- 
alcohol  is  not  a  sufficiently  strong  oxidizing  agent.  He  therefore  suggests 
the  addition  of  a  drop  of  a  0.5  per  cent  solution  of  sodium  nitrite  to  the 
acid-alcohol  mixture  before  warming  on  the  water-bath.  Try  this 
modification  also. 

4.  Salkowski's  Test. — Render  5  c.c.  of  urine  alkaline  with  a  few 
drops  of  a  10  per  cent  sodium  carbonate  solution  and  add  a  10  per  cent 
solution  of  calcium  chloride,  drop  by  drop,  until  the  supernatant  fluid 
exhibits  the  normal  urinary  color  when  the  contents  of  the  test-tube 
are  thoroughly  mixed.  Filter  off  the  precipitate,  and  after  washing  it 
place  it  in  a  second  tube  with  95  per  cent  alcohol.  Acidify  the  alcohol 
with  hydrochloric  acid  and,  if  necessary,  shake  the  tube  to  bring  the 
precipitate  into  solution.  Heat  the  solution  to  boiling  and  observe  the 
appearance  of  a  green  color  which  changes  through  blue  and  violet  to 
red;  if  no  bile  is  present  the  solution  does  not  undergo  any  color  change. 
This  test  will  frequently  exhibit  greater  delicacy  than  Gmelin's  test. 
Steensma's  suggestions  mentioned  under  Huppert's  Reaction,  above, 
apply  in  connection  with  this  test  also. 

5.  Hammarsten's  Reaction. — To  about  5  c.c.  of  Hammarsten's 
reagent^  in  a  small  evaporating  dish  add  a  few  drops  of  urine.  A  green 
color  is  produced.  If  more  of  the  reagent  is  now  added  the  play  of 
colors  as  noted  in  Gmelin  's  test  may  be  obtained. 

b.  Smith's  Test. — To  2-3  c.c.  of  urine  in  a  test-tube  add  carefully 
about  5  c.c.  of  dilute  tincture  of  iodine  (i :  10)  so  that  the  fluids  do  not 
mix.     A  green  ring  is  observed  at  the  point  of  contact. 

7.  Salkowski-Schippers  Reaction. — Neutralize  the  acidity  of  10 
c.c.  of  the  urine  under  examination  with  a  few  drops  of  a  dilute  solution 
of  sodium  carbonate,  and  add  5  drops  of  a  20  per  cent  solution  of  sodium 
carbonate  and  10  drops  of  a  20  per  cent  solution  of  calcium  chloride. 
Filter  off  the  resultant  precipitate  upon  a  hardened  filter  paper  and  wash 
it  with  water.  Remove  the  precipitate  to  a  small  porcelain  dish,  add 
3  c.c.  of  an  acid-alcohol  mixture'  and  a  few  drops  of  a  dilute  solution 
of  sodium  nitrite  and  heat.  The  production  of  a  green  color  indicates 
the  presence  of  bile  pigments. 

8.  Bonanno's  Reaction.^ — Place  5-10  c.c.  of  the  urine  under 
examination  in  a  small  porcelain  evaporating  dish  and  add  a  few  drops 
of  Bonanno's  reagent.*     If  bile  is  present  an  emerald-green  color  will 

'  Hammarsten's  reagent  is  made  by  nuxing  i  volume  of  25  per  cent  nitric  acid  and  19 
volumes  of  25  per  cent  hydrochloric  acid  and  then  adding  i  volume  of  this  acid  mixture 
to__4  volumes  of  95  per  cent  alcohol. 

-  Made  by  adding  5  c.c.  of  concentrated  hydrochloric  acid  to  95  c.c.  of  96  per  cent  alcohol. 

^  II  Tommasi,  2,  Xo.  21. 

*  This  reagent  may  be  prepared  by  dissolving  2  grams  of  sodium  nitrite  in  100  c.c.  of 
concentrated  hvdrochloric  acid. 


344  PHYSIOLOGICAL   CHEMISTRY. 

develop.  Bonanno  says  the  reaction  is  not  interfered  with  by  any  known 
normal  or  pathological  urinary  constituent. 

Tests  for  Bile  Acids. 

1.  Pettenkofer's  Test. — To  5  c.c.  of  urine  in  a  test-tube  add  5 
drops  of  a  5  per  cent  solution  of  sucrose.  Now  incline  the  tube,  run 
about  2-3  c.c.  of  concentrated  sulphuric  acid  carefully  down  the  side 
and  note  the  red  ring  at  the  point  of  contact.  Upon  slightly  agitating 
the  contents  of  the  tube  the  whole  solution  gradually  assumes  a  reddish 
color.  As  the  tube  becomes  warm,  it  should  be  cooled  in  running  water 
in  order  that  the  temperature  may  not  rise  above  70°  C. 

2.  Mylius's  Modification  of  Pettenkofer's  Test. — To  approxi- 
mately 5  c.c.  of  urine  in  a  test-tube  add  3  drops  of  a  very  dilute  (i :  1000) 
aqueous  solution  of  furfurol, 

HC CH 

HC         C.CHO. 

O 

Now  incline  the  tube,  run  about  2-3  c.c.  of  concentrated  sulphuric  acid 
carefully  down  the  side  and  note  the  red  ring  as  above.  In  this  case 
also,  upon  shaking  the  tube,  the  whole  solution  is  colored  red.  Keep 
the  temperature  below  70°  C.  as  before. 

3.  Neukomm's  Modification  of  Pettenkofer's  Test. — To  a  few 
drops  of  urine  in  an  evaporating  dish  add  a  trace  of  a  dilute  sucrose 
solution  and  one  or  more  drops  of  dilute  sulphuric  acid.  Evaporate 
on  a  water-bath  and  observe  the  development  of  a  violet  color  at  the 
edge  of  the  evaporating  mixture.  Discontinue  the  evaporation  as  soon 
as  the  color  is  observed. 

4.  V.  Udransky's  Test. — To  5  c.c.  of  urine  in  a  test-tube  add  3-4 
drops  of  a  very  dilute  (i :  1000)  aqueous  solution  of  furfurol.  Place 
the  thumb  over  the  top  of  the  tube  and  shake  until  a  thick  foam  is  formed. 
By  means  of  a  small  pipette  add  2-3  drops  of  concentrated  sulphuric 
acid  to  the  foam  and  observe  the  dark  pink  coloration  produced. 

5.  Hay's  Test. — This  test  is  based  upon  the  principle  that  bile  acids 
have  the  property  of  reducing  the  surface  tension  of  fluids  in  which 
they  are  contained.  The  test  is  performed  as  follows:  Cool  about  10  c.c. 
of  urine  in  a  test-tube  to  17°  C.  or  lower,  and  sprinkle  a  little  finely 
pulverized  sulphur  upon  the  surface  of  the  fluid.  The  presence  of  bile 
acids  is  indicated  if  the  sulphur  sinks  to  the  bottom  of  the  liquid,  the 
rapidity  with  which  the  sulphur  sinks  depending  upon  the  amount  of 
bile  acids  present  in  the  urine.  The  test  is  said  to  react  with  bile  acids 
when  the  latter  are  present  in  the  proportion  i :  120,000. 


URINE.  345 

Some  investigators  claim  that  it  is  impossible  to  differentiate  between 
bile  acids  and  bile  pigments  by  this  test. 

CH3 
ACETONE,        C  =  0. 

I 

CH, 

It  was  formerly  very  generally  believed  that  acetone  appeared  in 
the  urine  under  pathological  conditions  because  of  increased  protein 
decomposition.  It  is  now  generally  thought  that,  in  man,  the  output 
of  acetone  arises  principally  from  the  breaking  down  of  fatty  tissues 
or  fatty  foods  within  the  organism.  The  quantity  of  acetone  elimi- 
nated has  been  shown  to  increase  when  the  subject  is  fed  an  abundance 
of  fat-containing  food  as  well  as  during  fasting,  whereas  a  replace- 
ment of  the  fat  with  carbohydrates  is  followed  by  a  marked  decrease 
in  the  acetone  excretion.  Conditions  are  different  with  certain  of  the 
lower  animals.  With  the  dog,  for  instance,  the  output  of  acetone  is 
not  diminished  when  the  animal  is  fed  upon  a  carbohydrate  diet,  is 
decreased  during  fasting,  and  increased  when  the  animal  is  fed  upon 
a  diet  of  meat. 

Acetone  and  the  closely  related  bodies,  /9-oxybutyric  acid  and  dia- 
cetic  acid,  are  generally  classified  as  the  acetone  bodies.  They  are  all 
associated  with  a  deranged  metabolic  function  and  may  appear  in  the 
urine  together  or  separately,  depending  upon  the  conditions.  Acetone 
and  diacetic  acid  may  occur  alone  in  the  urine  but  ;9-oxybutyric  acid 
is  never  found  except  in  conjunction  with  one  or  the  other  of  these  bodies. 
Acetone  and  diacetic  acid  arise  chiefly  from  the  oxidation  of  ,5-oxybutyric 
acid.  The  relation  existing  between  these  three  bodies  is  shown  in  the 
following  reactions: 

(a)  CH3.CH(OH).CH.,.COOH  +  0--CH3CO.CH,.COOH-HH,0. 

^-oxybutyric  acid.  Diacetic  acid 

ih)  CH3CO.CH,.COOH->(CH3)3CO  +  CO,. 

Diacetic  acid.  Acetone. 

Acetone,  chemically  considered,  is  a  ketone,  di-methyl  ketone.  When 
pure  it  is  a  liquid  which  possesses  a  characteristic  aromatic  fruit-like 
odor,  boils  at  56-57°  C.  and  is  miscible  with  water,  alcohol,  or  ether 
in  all  proportions.  Acetone  is  a  physiological  as  well  as  a  pathological 
constituent  of  the  urine  and  under  normal  conditions  the  daily  output 
is  about  o.or-0.03  gram. 

Pathologically,  the  elimination  of  acetone  is  often  greatly  increased 
and  at  such  times  a  condition  of  acetonuria  is  said  to  exist.  This  patho- 
logical acetonuria  may  accompany  diabetes  mellitus,  scarlet  fever,  typhoid 


346  PHYSIOLOGICAL    CHEMISTRY. 

fever,  pneumonia,  nephritis,  phosphorus  poisoning,  grave  anaemias, 
fasting,  and  a  deranged  digestive  function;  it  also  frequently  accom- 
panies auto-intoxication  and  chloroform  and  ether  ansesthesia.  The 
types  of  acetonuria  most  frequently  met  with  are  those  noted  in  febrile 
conditions  and  in  advanced  cases  of  diabetes  mellitus. 

Experiments. 

1.  Isolation  from  the  Urine. — In  order  to  facilitate  the  detection 
of  acetone  in  the  urine,  the  specimen  under  examination  should  be 
distilled  and  the  tests  as  given  below  applied  to  the  resulting  distillate. 
If  it  is  not  convenient  to  distil  the  urine,  the  tests  may  be  conducted 
upon  the  undistilled  fluid.  To  obtain  an  acetone  distillate  proceed 
as  follows:  Place  100-250  c.c.  of  urine  in  a  distillation  flask  or  retort 
and  render  it  acid  with  acetic  acid.  Collect  about  one-third  of  the  orig- 
inal volume  of  fluid  as  a  distillate,  add  5  drops  of  10  per  cent  hydro- 
chloric acid  and  redistil  about  one-half  of  this  volume.  With  this  final 
distillate  conduct  the  tests  as  given  below. 

2.  Gunning's  lodofomi  Test. — To  about  5  c.c.  of  the  urine  or 
distillate  in  a  test-tube  add  a  few  drops  of  Lugol's  solution^  or  ordi- 
nary iodine  solution  (I  in  KI)  and  enough  NH^OH  to  form  a  black 
precipitate  (nitrogen  iodide).  Allow  the  tube  to  stand  (the  length  of 
time  depending  upon  the  content  of  acetone  in  the  fluid  under  examina- 
tion) and  note  the  formation  of  a  yellowish  sediment  consisting  of  iodo- 
form. Examine  the  sediment  under  the  microscope  and  compare  the 
form  of  the  crystals  with  those  shown  in  Fig.  7,  p.  47.  If  the  crystals  are 
not  well  formed  recrystallize  them  from  ether  and  examine  again.  The 
crystals  of  iodoform  should  not  be  confounded  with  those  of  stellar  phos- 
phate (Fig.  81,  p.  242)  which  mayb  e  formed  in  this  test,  particularly 
if  made  upon  the  undistilled  urine.  This  test  is  preferable  to  Lieben's 
test  (4)  since  no  substance  other  than  acetone  will  produce  iodoform 
when  treated  according  to  the  directions  for  this  test;  both  alcohol  and 
aldehyde  yield  iodoform  when  tested  by  Lieben's  test. 

Gunning's  test  is  rather  the  most  satisfactory  test  yet  suggested 
for  the  detection  of  acetone,  and  may  be  used  with  good  results  even 
upon  the  undistilled  urine.  In  some  instances  where  the  amount  of 
acetone  present  is  very  small  it  is  necessary  to  allow  the  tube  to  stand 
24  hours  before  making  the  examination  for  iodoform  crystals.  This 
test  serves  to  detect  acetone  when  present  in  the  ratio  i :  100,000. 

3.  Legal's  Test. — Introduce  about  5  c.c.  of  the  urine  or  distillate 
into  a  test-tube,  add  a  few  drops  of  freshly  prepared  aqueous  solution 

'  Lugol's  solution  may  be  prepared  by  dissolving  4  grams  of  iodine  and  6  grams  of  potas- 
sium iodide  in  100  c.c.  of  distilled  water. 


URINE.  347 

of  sodium  nitroprussidc  and  render  the  mixture  alkaline  with  potassium 
hydroxide.  A  ruby  red  color,  due  to  creatinine,  a  normal  urinary 
constituent,  is  produced  (see  Wcyl's  test,  p.  296).  Add  an  excess  of 
acetic  acid  and  if  acetone  is  present  the  red  color  will  be  intensified, 
whereas  in  the  absence  of  acetone  a  yellow  color  will  result.  Make 
a  control  test  upon  normal  urine  to  show  that  this  is  so.  A  similar  red 
color  may  be  produced  by  paracrcsol  in  urines  containing  no  acetone. 

4.  Lieben's  Test. — Introduce  5  c.c.  of  the  urine  or  distillate  into 
a  test-tube,  render  it  alkaline  with  potassium  hydroxide  and  add  1-2 
c.c.  of  iodine  solution  drop  by  drop.  If  acetone  is  present  a  yellowish 
precipitate  of  iodoform  will  be  produced.  Identify  the  iodoform  by 
means  of  its  characteristic  odor  and  its  typical  crystalline  form  (see 
Fig.  7,  p.  47).  While  fully  as  delicate  as  Gunning's  test  (2)  this  test 
is  not  as  accurate  since  by  means  of  the  procedure  involved,  either  alcohol 
or  aldehyde  will  yield  a  precipitate  of  iodoform.  This  test  is  especially 
liable  to  lead  to  erroneous  deductions  when  urines  from  the  advanced 
stages  of  diabetes  are  under  examination,  because  of  the  presence  of 
alcohol  formed  from  the  sugar  through  fermentative  processes.^ 

5.  Reynolds-Gunning  Test.— This  test  depends  upon  the  solu- 
bility of  mercuric  oxide  in  acetone  and  is  performed  as  follows:  To 
5  c.c.  of  the  urine  or  distillate  add  a  few  drops  of  mercuric  chloride, 
render  the  solution  alkaline  with  potassium  hydroxide  and  add  an  equal 
volume  of  95  per  cent  alcohol.  Shake  thoroughly  in  order  to  bring  the 
major  portion  of  the  mercuric  oxide  into  solution  and  filter.  Render  the 
clear  filtrate  faintly  acid  with  hydrochloric  acid  and  stratify  some  am- 
monium sulphide,  (NHJ2S,  upon  this  acid  solution.  At  the  zone  of 
contact  a  blackish-gray  ring  of  precipitated  mercuric  sulphide,  HgS, 
will  form.  Aldehyde  also  responds  to  this  test.  Aldehyde,  however, 
has  never  been  detected  in  the  urine  and  could  only  be  present  in  this 
instance  if  the  acidified  urine  was  distilled  too  far. 

6.  Taylor's  Test.— To  10  c.c.  of  the  urine  or  distillate  in  a  test-tube 
add  a  few  drops  of  a  freshly  prepared  aqueous  solution  of  sodium  nitro- 
prussidc and  stratify  concentrated  ammonium  hydroxide  upon  the  mixture. 
The  production  of  a  magenta  color  at  the  point  of  contact  indicates  the 
presence  of  acetone  in  the  urine  or  distillate  under  examination.  Normal 
urine  yields  an  orange-red  color  when  subjected  to  this  technic. 

Rothera's  Reaction.^ — To  5-10  c.c.  of  urine  or  distillate  in  a  test- 
tube  add  a  little  solid  ammonium  sulphate,  2-3  drops  of  a  freshly  pre- 

'  W'elker  reports  the  production  of  a  pink  or  red  color  during  the  application  of  this  test 
to  the  distillates  from  pathological  urines  which  had  been  preserved  with  powdered  thymol. 
He  found  the  color  to  be  due  to  an  iodothymol  compound  which  had  been  previously  prepared 
synthetically  by  Messinger  and  Vortmann. 

-  Rothera:  Jour.  Physiol.,  37,  491,  1908. 


348  PHYSIOLOGICAL   CHEMISTRY, 

pared  5  per  cent  solution  of  sodium  nitroprusside  and  1-2  c.c.  of  con- 
centrated ammonium  hydroxide.  The  development  of  a  permanganate 
color  indicates  the  presence  of  acetone. 

CH3 

I 
DIACETIC  ACID,  C  =  O 

CH2.COOH. 

Diacetic  or  acetoacetic  acid  occurs  in  the  urine  only  under  path- 
ological conditions  and  is  rarely  found  except  associated  with  acetone. 
It  is  formed  from  /?-oxybutyric  acid,  another  of  the  acetone  bodies,  and 
upon  decomposition  yields  acetone  and  carbon  dioxide.  Diaceturia 
occurs  ordinarily  under  the  same  conditions  as  the  pathological  ace- 
tonuria,  i.  e.,  in  fevers,  diabetes,  etc.  (see  p.  345).  If  very  little  diacetic 
acid  is  formed  it  may  be  transformed  into  acetone,  whereas  if  a  larger 
quantity  is  produced  both  acetone  and  diacetic  acid  may  be  present  in  the 
urine.  Diaceturia  is  most  frequently  observed  in  children,  especially 
accompanying  fevers  and  digestive  disorders;  it  is  perhaps  less  fre- 
quently observed  in  adults,  but  when  present,  particularly  in  fevers  and 
diabetes,  it  is  frequently  followed  by  fatal  coma. 

Diacetic  acid  is  a  colorless  liquid  which  is  miscible  with  water,  alcohol, 
and  ether,  in  all  proportions.  It  differs  from  acetone  in  giving  a  violet- 
red  or  Bordeaux-red  color  with  a  dilute  solution  of  ferric  chloride. 

Experiments. 

I.  Gerhardt's  Test.- — To  5  c.c.  of  urine  in  a  test-tube  add  ferric 
chloride  solution,  drop  by  drop,  until  no  more  precipitate  forms.  In 
the  presence  of  diacetic  acid  a  Bordeaux-red  color  is  produced;  this 
color  may  be  somewhat  masked  by  the  precipitate  of  ferric  phosphate, 
in  which  case  the  fluid  should  be  filtered. 

A  positive  result  from  the  above  manipulation  simply  indicates  the 
possible  presence  of  diacetic  acid.  Before  making  a  final  decision  re- 
garding the  presence  of  this  body  make  the  two  following  control  experi- 
ments: 

(a)  Place  5  c.c.  of  urine  in  a  test-tube  and  boil  it  vigorously  for 
3-5  minutes.  Cool  the  tube  and,  with  the  boiled  urine,  make  the  test 
as  given  above.  As  has  been  already  stated,  diacetic  acid  yields  acetone 
upon  decomposition  and  acetone  does  not  give  a  Bordeaux-red  color 
with  ferric  chloride.  By  boiling  as  indicated  above,  therefore,  any 
diacetic  acid  present  would  be  decomposed  into  acetone  and  carbon 
dioxide  and  the  test  upon  the  resulting  fluid  would  be  negative.  If 
positive  the  color  is  due  to  the  presence  of  bodies  other  than  diacetic  acid. 


URINE.  349 

(b)  Place  5  c.c.  of  urine  in  a  test-tube,  acidify  with  H2SO^,  to  free 
diacetic  acid  from  its  salts,  and  carefully  extract  the  mixture  with  ether 
by  shaking.  If  diacetic  acid  is  prcsnte  it  will  be  extracted  by  the  ether. 
Now  remove  the  ethereal  solution,  evaporate  it  to  dryness,  dissolve  the 
residue  in  1-2  c.c.  of  water  and  add  3-5  drops  of  3  per  cent  ferric  chloride. 
Diacetic  acid  is  indicated  by  the  production  of  the  characteristic  Bordeaux- 
red  color.  This^color  disappears  spontaneously  in  24-48  hours.  Such 
substances  as  antipyrin,  kairin,  phenacetin,  salicylic  acid,  salicylates, 
sodium  acetate,  thiocyanates,  and  thallin  yield  a  similar  red  color  under 
these  conditions,  but  when  due  to  the  presence  of  any  of  these  substances 
the  color  does  not  disappear  spontaneously  but  may  remain  permanent 
for  days.  Many  of  these  disturbing  substances  are  soluble  in  benzene  or 
chloroform  and  may  be  removed  from  the  urine  by  this  means  before 
extracting  with  ether  as  above.  Diacetic  acid  is  insoluble  in  benzene  or 
chloroform. 

2.  Arnold-Lipliawsky  Reaction.^ — This  reaction  is  somewhat  more 
delicate  than  Gerhardt's  test  (i)  and  serves  to  detect  diacetic  acid  when 
present  in  the  proportion  of  1:25,000.  It  is  also  negative  toward  acetone, 
^^-oxybutyric  acid  and  the  interfering  drugs  mentioned  as  causing  errone- 
ous deductions  in  the  application  of  Gerhardt's  test.  If  the  urine  under 
examination  is  highly  pigmented  it  should  be  partly  decolorized  by  means 
of  animal  charcoal  before  applying  the  test  as  indicated  below. 

Place  5  c.c.  of  the  urine  under  examination  and  an  equal  volume 
of  the  Arnold-Lipliawsky  reagent^  in  a  test-tube,  add  a  few  drops  of  con- 
centrated ammonia  and  shake  the  tube  vigorously.  Note  the  production 
of  a  brick-red  color.  Take  1-2  c.c.  of  this  colored  solution,  add  10-20 
c.c.  of  hydrochloric  acid  (sp.  gr.  i.ig),  3  c.c.  of  chloroform,  and  2-4 
drops  of  ferric  chloride  solution  and  carefully  mix  the  fluids.  Diacetic 
acid  is  indicated  by  the  chloroform  assuming  a  violet  or  blue  color;  if 
diacetic  acid  is  absent  the  color  may  be  yellow  or  light  red. 

H    OHH 

I     !     I 

^-OXYBUTYRIC  ACID,  H-C-C-C-  COOH. 

I         I        I 

H    H    H 

This  acid  does  not  occur  as  a  normal  constituent  of  urine  but  is  found 
only  under  pathological  conditions  and  then  always  in  conjunction  with 

'  This  reagent  consists  of  two  definite  solutions  which  are  ordinarily  preserved  separately 
and  mixed  just  before  using.     The  two  solutions  are  prepared  as  follows: 

(o)   One  per  cent  aqueous  solution  of  potassium  nitrite. 

(b)  (Jne  gram  of />-amino-acetophenon  dissolved  in  loo  c.c.  of  distilled  water  and  enough 
hydrochloric  acid  (about  2  c.c.)  added,  drop  by  drop,  to  cause  the  solution,  which  is  at  first 
yellow,  to  become  entirely  colorless.     An  excess  of  acid  must  be  avoided. 

Before  using,  a  and  b  are  mixed  in  the  ratio  1:2. 


350  PHYSIOLOGICAL    CHEMISTRY. 

either  acetone  or  diacetic  acid.  Either  of  these  bodies  may  be  formed 
from  /3-oxybutyric  acid  under  proper  conditions.  It  is  present  in  espe- 
cially large  amount  in  severe  cases  of  diabetes  and  has  also  been  detected 
in  digestive  disturbances,  continued  fevers,  scurvy,  measles,  and  in 
starvation.  It  is  probable  that,  in  man,  /?-oxybutyric  acid,  in  common 
with  acetone  and  diacetic  acid,  arises  principally  from  the  breaking  down 
of  fatty  tissues  within  the  organism.  The  condition  in  which  large 
amounts  of  acetone  and  diacetic  acid,  and  in  severe  cases  /?-oxybutyric 
acid  also,  are  excreted  in  the  urine  is  known  as  "acidosis."  In  diabetes 
the  deranged  metabolic  conditions  cause  the  production  of  great  quantities 
of  these  substances  which  lead  to  an  acid  intoxication  and  ultimately  to 
diabetic  coma. 

Ordinarily  /9-oxybutyric  acid  is  an  odorless,  transparent  syrup,  which 
is  laevorotatory  and  easily  soluble  in  water,  alcohol,  and  ether;  it  may  be 
obtained  in  crystalline  form. 

EXPEEIMENTS. 

I.  Black's  Reaction.^ — -Inasmuch  as  the  urinary  pigments  as  well  as 
any  contained  sugar  or  diacetic  acid  will  interfere  with  the  delicacy  of  this 
test  when  applied  to  the  urine  directly  the  following  preliminary  procedure 
is  necessary:  Concentrate  lo  c.c.  of  the  urine  under  examination  to  one- 
third  or  one-fourth  of  its  original  volume  in  an  evaporating  dish  at  a 
gentle  heat.  Acidify  the  residue  with  a  few  drops  of  concentrated  hydro- 
chloric acid,  add  sufficient  plaster  of  Paris  to  make  a  thick  paste  and  allow 
the  mixture  to  stand  until  it  begins  to  "set."  It  should  now  be  stirred 
and  broken  up  in  the  dish  by  means  of  a  stirring  rod  with  a  blunt  end. 
Extract  the  porous  meal  thus  produced  twice  with  ether  by  stirring  and 
decantation.  Any  ^S-oxybutyric  acid  present  will  be  extracted  by  the 
ether.  Evaporate  the  ether  extract  spontaneously  or  on  a  water-bath, 
dissolve  the  residue  in  water,  and  neutralize  it  with  barium  carbonate. 
To  5  to  lo  c.c.  of  this  neutral  fluid  in  a  test-tube  add  two  to  three  drops  of 
ordinary  commercial  acid  hydrogen  peroxide.  Mix  by  shaking  and  add  a 
few  drops  of  Black's  reagent.^  Permit  the  tube  to  stand  and  note  the 
gradual  development  of  a  rose  color  which  increases  to  its  maximum 
intensity  and  then  gradually  fades. ^ 

In  carrying  out  the  test  care  should  be  taken  to  see  that  the  solution  is 
cold  and  approximately  neutral  and  that  a  large  excess  of  hydrogen  peroxide 
and  Black's  reagent  are  not  added.  In  case  but  little  /?-oxybutyric  acid  is 
present  the  color  will  fail  to  appear  or  will  be  but  transitory  if  the  oxidizing 

'  Made  by  dissolving  5  grams  of  ferric  chloride  and  0.4  gram  of  ferrous  chloride  in  100 
c.c.  of  water. 

^  This  disappearance  of  color  is  due  to  the  further  oxirlation  of  the  diacetic  acid. 


URINE.  351 

agents  are  added  in  too  great  excess.  It  is  preferable  to  add  a  few  drops  of 
the  reagent  and  at  intervals  of  a  few  minutes  repeat  the  process  until  the 
color  undergoes  no  further  increase  in  intensity.  One  part  of  ^9-oxybutyric 
acid  in  10,000  parts  of  the  solution  may  be  detected  by  this  test. 

2,  Polariscopic  Examination. — Subject  some  of  the  urine  (free 
from  protein)  to  the  ordinary  fermentation  test  (see  page  331).  This  will 
remove  dextrose  and  laevulose,  which  would  interfere  with  the  polariscopic 
test.  Now  examine  the  fermented  fluid  in  the  polariscope  and  if  it  is 
laevorotatory  the  presence  of  /9-oxybutyric  acid  is  indicated.  This  test  is 
not  absolutely  reliable,  however,  since  conjugate  glycuronates  are  also 
lasvorotatory  after  fermentation. 

3.  Kulz's  Test. — Evaporate  the  urine,  after  fermenting  it  as  indicated 
in  the  last  test,  to  a  syrup,  add  an  equal  volume  of  concentrated  sulphuric 
acid,  and  distil  the  mixture  directly  without  cooling.  Under  these  con- 
ditions a-crotonic  acid  is  formed  and  is  present  in  the  distillate.  Allow 
the  distillate  to  cool  slowly  and  note  the  formation  of  crystals  of  a-crotonic 
acid  which  are  soluble  in  ether  and  melt  at  7  2°  C.  In  case  very  slight  traces 
of  /3-oxybutyric  acid  be  present  in  the  urine  under  examination  the  amount 
of  a-crotonic  acid  formed  may  be  too  small  to  yield  a  crystalline  product. 
In  this  event  the  distillate  should  be  extracted  with  ether,  the  ethereal 
extract  evaporated,  and  the  residue  washed  with  water.  Under  these 
conditions  the  impurities  will  be  removed  and  the  a-crotonic  acid  will 
remain  behind  as  a  residue.  The  melting-point  of  this  residue  may  then 
be  determined. 

CONJUGATE  GLYCURONATES. 

Glycuronic  acid  does  not  occur  free  in  the  urine,  but  is  found,  for  the 
most  part,  in  combination  with  phenol.  Much  smaller  quantities  are 
excreted  in  combination  with  indoxyl  and  skatoxyl.  The  total  content  of 
conjugate  glycuronates  seldom  exceeds  0.004  P^^  cent  under  normal 
conditions.  The  output  may  be  very  greatly  increased  as  the  result  of  the 
administration  of  antipyrin,  borneol,  camphor,  chloral,  menthol,  morphine, 
naphthol,  turpentine,  etc.  The  glycuronates  as  a  group  are  laevorotatory 
whereas  glycuronic  acid  is  dextro-rotatory.  Most  of  the  glycuronates, 
reduce  alkaline  metallic  oxides  and  so  introduce  an  error  in  the  examina- 
tion of  urine  for  sugar.  Conjugate  glycuronates  often  occur  associated 
with  dextrose  in,  glycosuria,  diabetes  mellitus,  and  in  some  other  disorders. 
As  a  class  the  glycuronates  are  non-fermentable. 

Experiments. 

I.  Fermentation-Reduction  Test. — Test  the  urine  by  Fehling's 
test.     If  there  is  reduction  try  Barfoed's  test.     If  negative  this  indicates 


352  PHYSIOLOGICAL    CHEMISTRY. 

the  absence  of  monosaccharides.  A  negative  fermentation  test  would 
now  indicate  the  presence  of  conjugate  glycuronates  (or  lactose  in  rare 
cases).  ^ 

If  dextrose  is  present  in  the  urine  tested  for  glycuronates  the  urine 
must  first  be  subjected  to  a  polariscopic  examination,  then  fermented 
and  a  second  polariscopic  examination  made.  The  sugar  being  dextro- 
rotatory and  fermentable  and  the  glycuronates  being  laevorotatory  and 
non-fermentable  the  second  polariscopic  test  will  show  a  laevorotation  in- 
dicative of  conjugate  glycuronates. 

2.  Tollens'  Reaction. — Make  this  test  according  to  directions  given 
under  Pentoses,  p.  353. 

PENTOSES. 

We  have  two  distinct  types  of  pentosuria,  i.  e.,  alimentary  pentosuria, 
resulting  from  the  ingestion  of  large  quantities  of  pentose-rich  vegetables 
such  as  prunes,  cherries,  grapes,  or  plums,  and  fruit  juices,  in 
which  condition  the  pentoses  appear  only  temporarily  in  the  urine; 
and  the  chronic  form  of  pentosuria,  in  which  the  output  of  pentoses  bears 
no  relation  whatever  to  the  quantity  and  nature  of  the  pentose  content  of 
the  food  eaten.  In  occurring  in  these  two  forms,  pentosuria  resembles 
glycosuria  (see  page  324),  but  it  is  definitely  known  that  pentosuria  bears 
no  relation  to  diabetes  mellitus  and  there  is  no  generally  accepted  theory 
to  account  for  the  occurrence  of  the  chronic  form  of  pentosuria.  The 
pentose  detected  most  frequently  in  the  urine  is  arabinose,  the  inactive 
form  generally  occurring  in  chronic  pentosuria  and  the  laevorotatory 

variety  occurring  in  the  alimentary  type  of  the  disorder. 

# 

Experiments. 

I.  Bial's  Reaction.- — To  5  c.c.  of  Bial's  reagent^  in  a  test-tube  add 
2-3  c.c.  of  urine  and  heat  the  mixture  gently  until  the  first  bubbles  rise  to 
the  surface.*  Immediately  or  upon  cooling  the  solution  becomes  green 
and  a  flocculent  precipitate  of  the  same  color  may  form. 

This  test  is  believed  to  be  more  accurate  than  the  orcinol  test.  It  is 
claimed  that  urines  containing  mew//io^,  kreosotal,  etc.,  respond  to  the  orcinol 
reaction,  but  not  to  Bial's. 

'  If  necessary  to  differentiate  Vjetween  lactose  and  glycuronates  apply  the  mucic  acid 
test  (see  p.  354)  or  the  phenylhydrazine  reaction  (see  p.  28). 
^  Bial:  Deut.  med.  Woch.,  28,  252,  i(;o2. 

'  Orcinol 1.5  gram. 

r'uming  HCI 500  grams. 

Ferric  chloride  do  per  cent) 20-30  drops. 

*  The  test  may  also  be  performed  by  adding  the  urine  to  the  hot  reagent.  No  further 
heating  should  be  necessary  if  pentose  is  present. 


URINE.  353 

2.  Tollens'  Reaction. — To  equal  volumes  of  urine  and  hydro- 
chloric acid  (sp.  gr.  1.09)  add  a  little  phloroglucinol  and  heat  the  mix- 
ture on  a  boiling  water-bath.  Pentose,  galactose,  or  glycuronic  acid  will 
be  indicated  by  the  appearance  of  a  red  color.  To  differentiate  between 
these  bodies  examine  by  the  spectroscope  and  look  for  the  absorption 
band  between  D  and  E  given  by  pentoses  and  glycuronic  acid,  and  then 
differentiate  between  the  two  latter  bodies  by  the  melting-points  of  their 
osazones. 

3.  Orcinol  Test. — Place  equal  volumes  of  urine  and  hydrochloric 
acid  (sp.  gr.  1.09)  in  a  test-tube,  add  a  small  amount  of  orcinol,  and 
heat  the  mixture  to  boiling.  Color  changes  from  red  through  reddish- 
blue  to  green  will  be  noted.  When  the  solution  becomes  green  it  should 
be  shaken  in  a  separatory  funnel  with  a  little  amyl  alcohol,  and  the  alco- 
holic extract  examined  spectroscopically.  An  absorption  band  between 
C  and  D  will  be  observed. 

FAT. 

When  fat  finds  its  way  into  the  urine  through  a  lesion  which  brings 
some  portion  of  the  urinary  passages  into  communication  with  the  lym- 
phatic system  a  condition  known  as  chyluria  is  established.  The  turbid 
or  milky  appearance  of  such  urine  is  due  to  its  content  of  chyle.  This 
disease  is  encountered  most  frequently  in  tropical  countries,  but  is  not 
entirely  unknown  in  more  temperate  climates.  Albumin  is  a  constant 
constituent  of  the  urine  in  chyluria.  Upon  shaking  a  chylous  urine 
with  ether  the  fat  is  dissolved  by  the  ether  and  the  urine  becomes  clearer 
or  entirely  clear. 

HiEMATOPORPHYRIN. 

Urine  containing  this  body  is  occasionally  met  with  in  various  diseases, 
but  more  frequently  after  the  use  of  quinine,  tetronal,  trional,  and  espe- 
cially sulphonal.  Such  urines  ordinarily  possess  a  reddish  tint,  the  depth 
of  color  varying  greatly  under  different  conditions. 

Experiments. 

I.  Spectroscopic  Examination. — To  100  c.c.  of  urine  add  about 
20  c.c.  of  a  10  per  cent  solution  of  potassium  hydroxide  orammonium 
hydroxide.  The  precipitate  which  forms  consists  principally  of  earthy 
phosphates  to  which  the  haematoporphyrin  adheres  and  is  carried  down. 
Filter  off  the  precipitate,  wash  it  and  transfer  to  a  flask  and  warm  with 
23 


354  PHYSIOLOGICAL    CHEMISTRY. 

alcohol  acidified  with  hydrochloric  acid.  By  this  process  the  haematopor- 
phyrin  is  dissolved  and  on  filtering  will  be  found  in  the  filtrate  and  may  be 
identified  by  means  of  the  spectroscope  (see  page  219,  and  Absorption 
Spectra,  Plate  II). 

2.  Acetic  Acid  Test. — To  100  c.c.  of  urine  add  5  c.c.  of  glacial 
acetic  acid  and  allow  the  mixture  to  stand  48  hours.  Haematoporphyrin 
deposits  in  the  form  of  a  precipitate. 

LACTOSE. 

Lactose  is  rarely  found  in  the  urine  except  as  it  is  excreted  by  women 
during  pregnancy,  during  the  nursing  period,  or  soon  after  weaning. 
It  is  rather  difficult  to  show  the  presence  of  lactose  in  the  urine  in  a  satis- 
factory manner,  since  the  formation  of  the  characteristic  lactosazone  is 
not  attended  with  any  great  measure  of  success  under  these  conditions. 
It  is,  however,  comparatively  easy  to  show  that  it  is  not  dextrose,  for, 
while  it  responds  to  reduction  tests,  it  does  not  ferment  with  pure  yeast 
and  does  not  give  a  dextrosazone.  An  absolutely  conclusive  test,  of 
course,  is  the  isolation  of  the  lactose  in  crystalline  form  (Fig.  80,  p.  238) 
from  the  urine. 

On  oxidation  with  nitric  acid  lactose  and  galactose  yield  mucic  acid. 
This  test  is  frequently  used  in  urine  examination  to  differentiate  lactose 
and  galactose  from  other  reducing  sugars. 

Experiments. 

1.  Mucic  Acid  Test. — Treat  100  c.c.  of  the  urine  under  examination 
with  20  c.c.^  of  concentrated  nitric  acid  and  evaporate  the  mixture  in  a 
broad,  shallow  glass  vessel,  upon  a  boiling  water-bath  until  the  volume 
of  the  solution  is  only  about  20  c.c.  At  this  point  the  fluid  should  be 
clear  and  a  fine  white  precipitate  of  mucic  acid  should  separate.  If  the 
percentage  of  lactose  in  the  urine  is  low  it  may  be  necessary  to  cool  the 
solution  and  permit  it  to  stand  for  some  time  before  the  precipitate  will 
form.  It  is  impossible  to  differentiate  between  galactose  and  lactose  by 
means  of  this  test,  but  the  reaction  does  serve  to  differentiate  these  two 
sugars  from  all  other  reducing  sugars.  A  satisfactory  differentiation 
between  lactose  and  galactose  may  be  made  by  means  of  Barfoed's 
test,  p.  33  T. 

2.  Rubner's  Test. — To  10  c.c.  of  urine  in  a  small  beaker  add  some 
lead  acetate,  in  substance,  heat  to  boiling,  and  add  NH^OH  until_^no 

*  If  the  specific  gravity  of  the  urine  is  1020  or  over  it  is  necessary  to  use  25-35  c.c.  of  nitric 
acid.  Under  these  conditions  the  mi.vture  should  be  evaporated  until  the  remaining  volume 
is  approximately  equivalent  to  that  of  the  nitric  acid  a  !dcd. 


URINE.  355 

more  precipitate  is  dissolved.  In  the  presence  of  lactose  a  brick-red  or 
rose-red  color  develops,  whereas  dextrose  gives  a  coffee-brown  color, 
maltose  a  light  yellow  color,  and  laevulose  no  color  at  all  under  the  same 
conditions. 

3.  Compound  Test. — Try  the  phenylhydrazine  test,  the  fermentation 
test,  and  Barfoed's  test  according  to  directions  given  under  Dextrose, 
pages  324,  and  331.  If  these  are  negative,  try  Nylander's  test,  page 
330.     If  this  last  test  is  positive,  the  presence  of  lactose  is  indicated. 

GALACTOSE. 

Galactose  has  occasionally  been  detected  in  the  urine,  and  in  particular 
in  that  of  nursing  infants  afflicted  with  a  deranged  digestive  function. 
Lactose  and  galactose  may  be  differentiated  from  other  reducing  sugars 
which  may  be  present  in  the  urine  by  means  of  the  mucic  acid  test.  This 
test  simply  consists  in  the  production  of  mucic  acid  through  oxidation  of 
the  sugar  with  nitric  acid. 

Experiments. 

1.  Mucic  Acid  Test. — Treat  too  c.c.  of  the  urine  under  examination 
with  20  c.c*  of  concentrated  nitric  acid  and  evaporate  the  mixture  in  a 
broad,  shallow  glass  vessel,  upon  a  boiling  water-bath,  until  the  volume  of 
the  solution  is  only  about  20  c.c.  At  this  point  the  fluid  should  be  clear 
and  a  fine,  white  precipitate  of  mucic  acid  should  separate.  If  the  per- 
centage of  galactose  present  in  the  urine  is  low  it  may  be  necessary  to  cool 
the  solution  and  permit  it  to  stand  for  some  time  before  the  precipitate 
will  form.  It  is  impossible  to  differentiate  between  galactose  and  lactose 
by  means  of  this  test,  but  the  reaction  does  serve  to  differentiate  these  two 
sugars  from  all  other  reducing  sugars.  A  satisfactory  differentiation 
between  galactose  and  lactose  may  be  made  by  Barfoed's  test,  p.  331. 

2.  Tollens'  Reaction. — To  equal  volumes  of  the  urine  and  hydro- 
chloric acid  (sp.  gr.  1.09)  add  a  little  phloroglucinol  and  heat  the  mixture 
on  a  boiling  water-bath.  Galactose,  pentose,  and  glycuronic  acid  will  be 
indicated  by  the  appearance  of  a  red  color.  Galactose  may  be  differen- 
tiated from  the  two  latter  substances  in  that  its  solutions  exhibit  no  absorp- 
tion bands  upon  spectroscopical  examination. 

LiEVULOSE. 

Diabetic  urine  frequently  possesses  the  power  of  rotating  the  plane  of 
polarized  light  to  the  left,  thus  indicating  the  presence  of  a  laevorotatory 

'  If  the  specific  gravity  of  the  urine  is  1020  or  over  it  is  necessary  to  use  25-35  ^•^-  oi 
nitric  acid.  Under  these  conditions  the  mixture  should  be  evaporated  until  the  remainin«^ 
volume  is  approximately  equivalent  to  that  of  the  nitric  acid  added. 


356  PHYSIOLOGICAL    CHEMISTRY, 

substance.  The  Isevorotation  is  sometimes  due  to  the  presence  of  laevu- 
lose,  although  not  necessarily  confined  to  this  carbohydrate,  since  conju- 
gate glycuronates  and  /3-oxybutyric  acid,  two  other  laevorotatory  bodies, 
are  frequently  found  in  the  urine  of  diabetics.  Laevulose  is  invariably 
accompanied  by  dextrose  in  diabetic  urine,  but  lavulosuria  has  been 
observed  as  a  separate  anomaly.  The  presence  of  laevulose  may  be 
inferred  when  the  percentage  of  sugar,  as  determined  by  the  titration 
method,  is  greater  than  the  percentage  indicated  by  the  polariscopic 
examination. 

Experiments. 

1.  Borchardt's  Reaction.— To  about  5  c.c.  of  urine  in  a  test-tube 
add  an  equal  volume  of  25  per  cent  hydrochloric  acid  and  a  few  crystals 
of  resorcinol.  Heat  to  boiling  and  after  the  production  of  a  red  color,  cool 
the  tube  under  running  water  and  transfer  to  an  evaporating  dish  or 
beaker.  Make  the  mixture  slightly  alkaline  with  solid  potassium  hy- 
droxide, return  it  to  a  test-tube,  add  2-3  c.c.  of  acetic  ether,  and  shake  the 
tube  vigorously.  In  the  presence  of  laevulose  the  acetic  ether  is  colored 
yellow. 

The  only  urinary  constituents  which  interfere  with  the  test  are  nitrites 
and  indican  and  these  interfere  only  when  they  are  simultaneously  present. 
Under  these  conditions,  the  urine  should  be  acidified  with  acetic  acid  and 
heated  to  boiling  for  one  minute  to  remove  the  nitrites.  In  case  the 
indican  content  is  very  large,  it  will  impart  a  blue  color  to  the  acetic  ether, 
thus  masking  the  yellow  color  due  to  laevulose.  When  such  urines  are  to 
be  examined,  the  indican  should  first  be  removed  by  Obermayer's  test 
(seep.  299).  The  chloroform  should  then  be  discarded,  the  acid-urine 
mixture  diluted  with  one-third  its  volume  of  water,  and  the  test  applied 
as  described  above.  The  urine  of  patients  who  have  ingested  santonin 
or  rhubarb  respond  to  the  test.  The  test  will  serve  to  detect  laevulose  when 
present  in  a  dilution  of  i  :2ooo,  i.  e.,  0.05  per  cent. 

2.  Seliwanoff's  Reaction. — To  5  c.c.  of  Seliwanoff's  reagent^  in  a 
test-tube  add  a  few  drops  of  the  urine  under  examination  and  heat  the 
mixture  to  boiling.  The  presence  of  laevulose  is  indicated  by  the  produc- 
tion of  a  red  color  and  the  separation  of  a  red  precipitate.  The  latter  may 
be  dissolved  in  alcohol  to  which  it  will  impart  a  striking  red  color. 

If  the  boiling  be  prolonged  a  similar  reaction  may  be  obtained  with 
urines  containing  dextrose.  This  has  been  explained^  in  the  case  of 
dextrose  as  due  to  the  transformation  of  the  dextrose  into  laevulose  by  the 
catalytic  action  of  the  hydrochloric  acid.     The  precautions  necessary  for 

'  Seliwanofl's  reagent   may  be  prepared  by  dissolving  0.05  gram  of  resorcinol  in  100  c.c. 
of^dilute  (i  :  2)  hydrochloric  acid. 
*  ^  Koenigsfeld:  Bioch.  Zeit.,  38,  311,  1912. 


URINE.  357 

a  positive  test  for  laevulose  are  as  follows:  The  concentration  of  the 
hydrochloric  acid  must  not  be  more  than  12  per  cent.  The  reaction  (red 
color)  and  the  precipitate  must  be  observed  after  not  more  than  20-30 
seconds  of  boiling.  Dextrose  must  not  be  present  in  amounts  exceeding 
2  per  cent.  The  precipitate  must  be  soluble  in  alcohol  with  a  bright  red 
color. 

3.  Phenylhydrazine  Test. — Make  the  test  according  to  directions 
under  Dextrose,  3,  page  324. 

4.  Polariscopic  Examination. — A  simple  polariscopic  examination, 
when  taken  in  connection  with  other  ordinary  tests,  will  furnish  the 
requisite  data  regarding  the  presence  of  laevulose,  provided  laevulose  is  not 
accompanied  by  other  laevorotatory  substances,  such  as  conjugate  gly- 
curonates  and  ,5-oxybutyric  acid. 

CHOH 


HOHC       CHOH 
INOSITE,  I  I 

HOHC       CHOH 

\/ 
CHOH 

Inosite  occasionally  occurs  in  the  urine  in  albuminuria,  diabetes 
mellitus,  and  diabetes  insipidus.  It  is  claimed  also  that  copious  water- 
drinking  causes  this  substance  to  appear  in  the  urine.  Inosite  was  at  one 
time  considered  to  be  a  sugar  but  is  now  known  to  be  hexahydroxybenzene, 
as  the  above  formula  indicates.  It  is  an  example  of  a  non-carbohydrate 
in  whose  molecule  the  H  and  O  are  present  in  the  proportion  to  form 
water.  In  other  words  it  has  the  formula  of  the  hexoses,  i.  e.,  CgHi^Og. 
Inosite  occurs  widely  distributed  in  the  vegetable  kingdom,  and  because 
of  this  fact  the  theory  has  been  voiced  that  it  represents  one  of  the  first 
stages  in  the  conversion  of  a  carbohydrate  into  the  benzene  ring.  It  is 
found  in  the  liver,  spleen,  lungs,  brain,  kidneys,  suprarenal  capsules, 
muscles,  leucocytes,  testes,  and  urine  under  normal  conditions. 

Experiment. 

I.  Detection  of  Inosite. — Acidify  the  urine  with  concentrated  nitric 
acid  and  evaporate  nearly  to  dryness.  Add  a  few  drops  of  ammonium 
hydroxide  and  a  little  calcium  chloride  solution  to  the  moist  residue  and 
evaporate  the  mixture  to  dryness.  In  the  presence  of  inosite  (0.00 1  gram) 
a  bright  red  color  is  obtained. 

For  a  more  satisfactory  test,  which  is  also  more  time-consuming,  see 
Salkowski's^  modification  of  Scherer's  test. 

'  Salkowski:  Zeit.  physiol.  client.,  69,  478,  1910. 


358  PHYSIOLOGICAL   CHEMISTRY. 

LAIOSE. 

This  substance  is  occasionally  found  in  the  urine  in  severe  cases  of 
diabetes  mellitus.  By  some  investigators  laiose  is  classed  with  the  sugars. 
It  resembles  laevulose  in  that  it  has  the  property  of  reducing  certain  metallic 
oxides  and  is  laevorotatory,  but  differs  from  laevulose  in  being  amorphous, 
non-fermentable,  and  in  not  possessing  a  sweet  taste. 

MELANINS. 

These  pigments  never  occur  normally  in  the  urine,  but  are  present 
under  certain  pathological  conditions,  their  presence  being  especially 
associated  with  melanotic  tumors.  Ordinarily  the  freshly  passed  urine  is 
clear,  but  upon  exposure  to  the  air  the  color  deepens  and  may  at  last  be 
very  dark  brown  or  black  in  color.  The  pigment  is  probably  present  in 
the  form  of  a  chromogen  or  melanogen  and  upon  coming  in  contact  with 
the  air  oxidation  occurs,  causing  the  transformation  of  the  melanogen 
into  melanin  and  consequently  the  darkening  of  the  urine. 

It  is  claimed  that  melanuria  is  proof  of  the  formation  of  a  visceral 
melanotic  growth.  In  many  instances,  without  doubt,  urines  rich  in 
indican  have  been  wrongly  taken  as  diagnostic  proof  of  melanuria.  The 
pigment  melanin  is  sometimes  mistaken  for  indigo  and  melanogen  for 
indican.  It  is  comparatively  easy  to  differentiate  between  indigo  and 
melanin  through  the  solubility  of  the  former  in  chloroform. 

In  rare  cases  melanin  is  found  in  urinary  sediment  in  the  form  of  fine 
amorphous  granules. 

Experiments. 

1.  Zeller's  Test. — To  50  c.c.  of  urine  in  a  small  beaker  add  an 
equal  volume  of  bromine  water.  In  the  presence  of  melanin  a  yellow 
precipitate  will  form  and  will  gradually  darken  in  color,  ultimately 
becoming  black. 

2.  von  Jaksch-PoUak  Reaction. — Add  a  few  drops  of  ferric 
chloride  solution  to  to  c.c.  of  urine  in  a  test-tube  and  note  the  formation 
of  a  gray  color.  Upon  the  further  addition  of  the  chloride  a  dark  precipi- 
tate forms,  consisting  of  phosphates  and  adhering  melanin.  An  excess  of 
ferric  chloride  causes  the  precipitate  to  dissolve. 

This  is  the  most  satisfactory  test  for  the  identification  of  melanin  in 
the  urine. 

UROROSEIN. 

This  is  a  pigment  which  is  not  present  in  normal  urine  but  may 
be  detected  in  the  urine  of  various  diseases,  such  as  pulmonary  tuber- 


URINE.  359 

culosis,  typhoid  fever,  nephritis,  and  stomach  disorders.  Urorosein,  in 
common  with  various  other  pigments,  does  not  occur  preformed  in  the 
urine,  but  is  present  in  the  form  of  a  chromogen,  which  is  transformed 
into  the  pigment  upon  treatment  with  a  mineral  acid. 

Experiments. 

1.  Robin's  Reaction. — -Acidify  lo  c.c.  of  urine  with  about  15  drops 
of  concentrated  hydrochloric  acid.  Upon  allowing  the  acidified  urine  to 
stand,  a  rose-red  color  will  appear  if  urorosein  is  present. 

2.  Nencki  and  Sieber's  Reaction. — To  100  c.c.  of  urine  in  a  beaker 
add  ID  c.c.  of  25  per  cent  sulphuric  acid.  Allow  the  acidified  urine 
to  stand  and  note  the  appearance  of  a  rose-red  color.  The  pigment  may 
be  separated  by  extraction  with  amyl  alcohol. 

UNKNOWN  SUBSTANCES. 

Ehrlich's  Diazo  Reaction. — Place  equal  volumes  of  urine  and 
Ehrlich's  diazobenzenesulphonic  acid  reagent^  in  a  test-tube,  mix  thor- 
oughly by  shaking,  and  quickly  add  ammonium  hydroxide  in  excess. 
The  test  is  positive  if  both  the  fluid  and  the  foam  assume  a  red  color. 
If  the  tube  is  allowed  to  stand  a  precipitate  forms,  the  upper  portion  of 
which  exhibits  a  blue,  green,  greenish-black,  or  violet  color.  Normal 
urine  gives  a  brownish-yellow  reaction  with  the  above  manipulation. 

The  exact  nature  of  the  substance  or  substances  upon  whose  pres- 
ence in  the  urine  this  reaction  depends  is  not  well  understood.  Some 
investigators  claim  that  a  positive  reaction  indicates  an  abnormal  de- 
composition of  protein  material,  whereas  others  assume  it  to  be  due 
to  an  increased  excretion  of  alloxyproteic  acid,  oxyproteic  acid,  or  uroferric 
acid. 

The  reaction  may  be  taken  as  a  metabolic  symptom  of  certain  dis- 
orders, which  is  of  value  diagnostically  otily  when  taken  in  connection 
with  the  other  symptoms.  The  reaction  appears  principally  in  the  urine 
in  febrile  disorders  and  in  particular  in  the  urine  in  typhoid  fever,  tubercu- 
losis, and  measles.  The  reaction  has  also  been  obtained  in  the  urine 
in   various   other   disorders   such   as   carcinoma,    chronic   rheumatism, 

'  Two  separate  solutions  should  be  prepared  and  mixed  in  definite  proportions  when 
needed  for  use. 

(o)  Five  grams  of  sodium  nitrite  dissolved  in  i  liter  of  distilled  water. 

(b)  Five  grams  of  sulphanilic  acid  and  50  c.c.  of  hydrochloric  acid  in  i  liter  of  distilled 
water. 

Solutions  a  and  b  should  be  preserved  in  well-stoppered  vessels  and  mixed  in  the  pro- 
portion I  :  50  when  required.  Green  asserts  that  greater  delicacy  is  secured  by  mixing  the 
solutions  in  the  proportion  i  :  100.  The  sodium  nitrite  deteriorates  upon  standing  and  be- 
comes unfit  for  use  in  the  course  of  a  few  weeks. 


360  PHYSIOLOGICAL   CHEMISTRY. 

diphtheria,  erysipelas,  pleurisy,  pneumonia,  scarlet  fever,  syphilis, 
typhus,  etc.  The  administration  of  alcohol,  chrysarobin,  creosote, 
cresol,  dionin,  guaiacol,  heroin,  morphine,  naphthalene,  opium,  phenol, 
tannic  acid,  etc.,  will  also  cause  the  urine  to  give  a  positive  reaction. 
The  following  chemical  reactions  take  place  in  this  test: 

(a)  NaNO^  +  HCl— HNO^  +  NaCl. 

NH,  N 

(b)  CeH,  +HNO,— CgH,  N  +  2H2O. 

\  \    / 

HSO3  SO3 

Sulphanilic  acid.  Diazo-benzenesulphonic  acid. 


CHAPTER  XX. 

URINE :  ORGANIZED  AND  UNORGANIZED 

SEDIMENTS. 

The  data  obtained  from  carefully  conducted  microscopical  exami- 
nations of  the  sediment  of  certain  pathological  urines  are  of  very  great 
importance,  diagnostically.  Too  little  emphasis  is  sometimes  placed 
upon  the  value  of  such  findings. 


Fig.  I02. — The  Purdv  Electric  Centrifuge. 


Fig.  103. — Sediment  Tube  for  the 
PuRDY  Electric  Centrifuge. 


The  sedimentary  constituents  may  be  divided  into  two  classes, 
i.  e.,  organized  and  unorganized.  The  sediment  is  ordinarily  collected 
for  examination  by  means  of  the  centrifuge  (Fig.  102,  above).  An  older 
method,  and  one  sill  in  vogue  in  some  quarters,  is  the  so-called  gravity 
method.  This  simply  consists  in  placing  the  urine  in  a  conical  glass 
and  allowing  the  sediment  to  settle.  The  collection  of  the  sediment  by 
means  of  the  centrifuge,  however,  is  much  preferable,  since  the  process 
of  sedimentation  may  be  accomplished  by  the  use  of  this  instrument  in  a 
few  minutes,  and  far  more  perfectly,  whereas  when  the  other  method  is 

361 


362  PHYSIOLOGICAL    CHEMISTRY. 

used  it  is  frequently  necessary  to  allow  the  urine  to  remain  in  the  con- 
ical glass  12-24  hours  before  sufficient  sediment  can  be  secured  for  the 
microscopical  examination. 

(a)  Unorganized  Sediments. 

Ammonium  magnesium  phosphate  (''Triple  phosphate"). 

Calcium  oxalate. 

Calcium  carbonate. 

Calcium  phosphate. 

Calcium  sulphate. 

Uric  acid. 

Urates. 

Cystine. 

Cholesterol. 

Hippuric  acid. 

Leucine  ■(  ?)  and  tyrosine. 

Haematoidin  and  biUrubin. 

Magnesium  phosphate. 

Indigo. 

Xanthine. 

Melanin. 

Ammonium  Magnesium  Phosphate  ("Triple  Phosphate"). — 
Crystals  of  "triple  phosphate"  are  a  characteristic  constituent  of  the 
sediment  when  alkaline  fermentation  of  the  urine  has  taken  place  either 
hejore  or  after  being  voided.  They  may  even  be  detected  in  amphoteric 
or  slightly  acid  urine  provided  the  ammonium  salts  are  present  in  large 
enough  quantity.  This  substance  may  occur  iil  the  sediment  in  two 
forms,  i.  e.,  prisms  and  the  feathery  type.  The  prismatic  form  of  crystals 
(Fig.  loi,  p.  319)  is  the  one  most  commonly  observed  in  the  sediment;  the 
feathery  form  (Fig.  loi.  p.  319)  predominates  when  the  urine  is  made 
ammoniacal  with  ammonia. 

The  sediment  of  the  urine  in  such  disorders  as  are  accompanied  by  a 
retention  of  urine  in  the  lower  urinary  tract  contains  "triple  phosphate" 
crystals  as  a  characteristic  constituent.  The  crystals  are  frequently 
abundant  in  the  sediment  during  paraplegia,  chronic  cystitis,  enlarged 
prostate,  and  chronic  pyelitis. 

Calcium  Oxalate. — Calcium  oxalate  is  found  in  the  urine  in  the  form 
of  at  least  two  distinct  types  of  crystals,  i.  e.,  the  dumb-bell  type  and  the 
octahedral  type  (Fig.  104,  p.  363).  Either  form  may  occur  in  the  sediment  of 
neutral,  alkaline,  or  acid  urine,  but  both  forms  are  found  most  frequently 
in  urine  having  an  acid  reaction.  Occasionally,  in  alkaline  urine,  the 
octahedral  form  is  confounded  with  "triple  phosphate"  crystals.     They 


URINE. 


;63 


may  be  differentiated  from  the  phosphate  crystals  by  the  fact  that  they 
are  insoluble  in  acetic  acid. 

The  presence  of  calcium  oxalate  in  the  urine  is  not  of  itself  a  sign  of  any 
abnormality,  since  it  is  a  constituent  of  normal  urine.  It  is  increased  above 
the  normal,  however,  in  such  pathological  conditions  as  diabetes  mellitus, 


» 


^ 


•  s 


^ 


% 


<^ 


9 


##       ♦ 


Fig.  104. — Calcium  Oxal.\te.     (Ogdeti.) 

in  organic  diseases  of  the  liver,  and  in  various  other  conditions  which  are 
accompanied  by  a  derangement  of  digestion  or  of  the  oxidation  mechan- 
ism, such  as  occurs  in  certain  diseases  of  the  heart  and  lungs. 

Calcium  Carbonate. — Calcium  carbonate  crystals  form  a  typical 
constituent  of  the  urine  of  herbivorous  animals.     Thev  occur  less  fre- 


FiG.  105. — Calcium  Carbox.a.te. 

quently  in  human  urine.  The  reaction  of  urine  containing  these  crystals 
is  nearly  always  alkaline,  although  they  may  occur  in  amphoteric  or  in 
slightly  acid  urine.  It  generally  crystallizes  in  the  form  of  granules, 
spherules,  or  dumb-bells  (Fig.  105,  above).  The  crystals  of  calcium 
carbonate  may  be  differentiated  from  calcium  oxalate  by  the  fact  that  they 
dissolve  in  acetic  acid  with  the  evolution  of  carbon  dioxide  gas. 


364  PHYSIOLOGICAL    CHEMISTRY. 

Calcium  Phosphate  (Stellar  Phosphate). — Calcium  phosphate  may 
occur  in  the  urine  in  three  forms,  i.  e.,  amorphous,  granular,  or  crystalline. 
The  crystals  of  calcium  phosphate  are  ordinarily  pointed,  wedge-shaped 
formations  which  may  occur  as  individual  crystals,  or  grouped  together 
in  more  or  less  regularly  formed  rosettes  (Fig.  81,  p.  242).  Acid  sodium 
urate  crystals  (Fig.  107,  p.  366)  are  often  mistaken  for  crystals  of  calcium 
phosphate.  We  may  differentiate  between  these  two  crystalline  forms  by 
the  fact  that  acetic  acid  will  readily  dissolve  the  phosphate,  whereas  the 
urate  is  much  less  soluble  and  when  finally  brought  into  solution  and  re- 
crystallized  one  is  frequently  enabled  to  identify  uric  acid  crystals  which 
have  been  formed  from  the  acid  urate  solution.  The  clinical  significance 
of  the  occurrence  of  calcium  phosphate  crystals  in  the  urinary  sediment 
is  similar  to  that  of  "triple  phosphate"  (see  page  319). 

Calciuni  Sulphate. — Crystals  of  calcium  sulphate  are  of  quite  rare 
occurrence  in  the  sediment  of  urine.  Their  presence  seems  to  be  limited 
in  general  to  urines  which  are  of  a  decided  acid  reaction.  Ordinarily 
it  crystallizes  in  the  form  of  long,  thin,  colorless  needles  or  prisms  (Fig.  100, 
page  316)  which  may  be  mistaken  for  calcium  phosphate  crystals.  There 
need  be  no  confusion  in  this  respect,  however,  since  the  sulphate  crystals 
are  insoluble  in  acetic  acid,  which  reagent  readily  dissolves  the  phosphate. 
As  far  as  is  known  their  oc  currence  as  a  constituent  of  urinary  sediment 
is  of  very  little  clinical  significance. 

Uric  Acid. — Uric  acid  forms  a  very  common  constituent  of  the  sedi- 
ment of  urines  which  are  acid  in  reaction.  It  occurs  in  more  varied  forms 
than  any  of  the  other  crystalline  sediments  (Plate  V,  opposite  page  291, 
and  Fig,  106,  page  365),  some  of  the  more  common  varieties  of  crystals 
being  rhombic  prisms,  wedges,  dumb-bells,  whetstones,  prismatic  rosettes, 
irregular  or  hexagonal  plates,  etc.  Crystals  of  pure  uric  acid  are  always 
colorless  (Fig.  94,  page  293),  but  the  form  occurring  in  urinary  sediments  is 
impure  and  under  the  microscope  appears  pigmented,  the  depth  of  color 
varying  from  light  yellow  to  a  dark  reddish-brown  according  to  the  size 
and  form  of  the  crystal. 

The  presence  of  a  considerable  uric  acid  sediment  does  not,  of  necessity, 
indicate  a  pathological  condition  or  a  urine  of  increased  uric  acid  content, 
since  this  substance  very  often  occurs  as  a  sediment  in  urines  whose  uric 
acid  content  is  diminished  from  the  normal  merely  as  a  result  of  changes  in 
reaction,  etc.  Pathologically,  uric  acid  sediments  occur  in  gout,  acute 
febrile  conditions,  chronic  interstitial  nephritis,  etc.  If  the  microscopical 
examination  is  not  conclusive,  uric  acid  may  be  differentiated  from  other 
crystalline  urinary  sediments  from  the  fact  that  it  is  soluble  in  alkalis, 
alkali  carbonates,  boiling  glycerol,  concentrated  sulphuric  acid,  and  in 
certain  organic  bases  such  as  ethylamine  and  piperidin.     It  also  responds 


PLATE  \-I. 


Ammontum  Urates,  showing  Spherules  and  Thorn-apple-shaped  Crystals. 
(From  Os^deii.  after  Peyer.) 


URINE. 


365 


to  the  murcxide  test  (see  page  292),  SchifT's  reaction  (see  page  293)  and 
to  Moreigne's  reaction  (see  p.   293). 

Urates. — The  urate  sediment  may  consist  of  a  mixture  of  the  urates  of 
ammonium,  calcium,  magnesium,  potassium,  and  sodium.  The  ammo- 
nium urate  may  occur  in  neutral,  alkaline,  or  acid  urine,  whereas  the  other 
forms  of  urates  are  confined  to  the  sediments  of  acid  urines.  Sodium 
urate  occurs  in  sediments  more  abundantly  than  the  other  urates.  There 
are  two  sodium  urates,  the  mono  and  the  di,  which  may  be  expressed  thus: 

Na+\^„  ,,  ^         ,  Na+^ 
j^  +  ^^tl2^4<-'3  and  ^^+ 


^C.H^N.O,. 


Both    salts    dissociate    with 
the  production  of  an  alkaline  reaction,  the  alkalinity  being  stronger  in  the 


Fig.  106. — Various  Forms  of  Uric  Acid. 
I,  Rhombic  plates;  2,  whetstone  forms;  3,  3,  quadrate  forms;  4,  5,  prolonged  into  points; 
6,  8,  rosettes;  7,  pointed  bundles;  9,  barrel  forms  precipitated  by  adding  hydrochloric  acid 
to  urine. 

case  of  the  di-sodium  urate.  The  so-called  quadriurate  or  hemiurate  have 
no  existence  as  chemical  units.  ^  The  urates  of  calcium,  magnesium,  and 
potassium  are  amorphous  in  character,  whereas  the  urate  of  ammonium 
is  crystalline.  Sodium  urate  may  be  either  amorphous  or  crystalline. 
When  crystalline  it  forms  groups  of  fan-shaped  clusters  or  colorless, 
prismatic  needles  (Fig.  107,  p.  366).  Ammonium  urate  is  ordinarily 
present  in  the  sediment  in  the  burr-like  form  of  the  "thorn-apple"  crystal, 
i.  e.,  yellow  or  reddish-brown  spheres,  covered  with  sharp  spicules  or 
prisms  (Plate  VI,  opposite).  The  urates  are  all  soluble  in  hydrochloric 
acid  or  acetic  acid  and  their  acid  solutions  yield  crystals  of  uric  acid  upon 
standing.     They  also  respond  to  the  murexide  test.     The  clinical  signifi- 

Taylor:  Jour.  Biol.  Chem.,  i,  177,  1905. 


;66 


PHYSIOLOGICAL    CHEMISTRY. 


cance  of  urate  sediments  is  very  similar  to  that  of  uric  acid.  A  considerable 
sediment  of  amorphous  urates  does  not  necessarily  indicate  a  high  uric 
acid  content,  but  ordinarily  signifies  a  concentrated  urine  having  a  very 
strong  acidity. 


Fig.  107. — Acid  Sodium  Urate. 

Cystine. — Cystine  is  one  of  the  rarer  of  the  crystalline  urinary  sedi- 
ments. It  has  been  claimed  that  it  occurs  more  often  in  the  urine  of  men 
than  of  women.  Cystine  crystallizes  in  the  form  of  thin,  colorless,  hexa- 
gonal plates  (Fig.  25,  p.  81,  and  Fig.  108,  below)  which  are  insoluble  in 
water,  alcohol,  and  acetic  acid,  and  soluble  in  minerals  acids,  alkalis, 
and  especially  in  ammonia.     Cystine  may  be  identified  by  burning  it  upon 


/ 


Fig.  108. — Cystine.     (Ogden.) 


platinum  foil,  under  which  condition  it  does  not  melt  but  yields  a  bluish- 
green  flame. 

Cholesterol. — Cholesterol  crystals  have  been  but  rarely  detected  in 
urinary  sediments.  When  present  they  probably  arise  from  a  pathological 
condition  of  some  portion  of  the  urinary  tract.  Crystals  of  cholesterol 
have  been  found  in  the  sediment  in  cystitis,  pyelitis,  chyluria,  and  nephritis. 


URINE.  367 

Ordinarily  it  crystallizes  in  large  regular  and  irregular  colorless,  transpar- 
ent plates,  some  of  which  possess  notched  corners  (Fig.  43,  page  166). 
Frecjuently,  instead  of  occurring  in  the  sediment,  it  is  found  in  the  form 
of  a  film  on  the  surface  of  the  urine. 

Hippuric  Acid. — This  is  one  of  the  rarer  sediments  of  human  urine. 
It  deposits  under  conditions  similar  to  those  which  govern  the  formation 
of  uric  acid  sediments.  The  crystals,  w^hich  are  colorless  needles  or  prisms 
(Fig.  97,  page  300)  when  pure,  are  invariably  pigmented  in  a  manner 
similar  to  the  uric  acid  crystals  when  observed  in  urinary  sediment  and 
because  of  this  fact  are  frequently  confounded  with  the  rarer  forms  of 
uric  acid.  Hippuric  acid  may  be  differentiated  from  uric  acid  from  the 
fact  that  it  does  not  respond  to  the  murexide  test  and  is  much  more  soluble 
in  water  and  in  ether.  The  detection  of  crystals  of  hippuric  acid  in 
the  urine  has  very  little  clinical  significance,  since  its  presence  in  the 
sediment  depends  in  most  instances  very  greatly  upon  the  nature  of 
the  diet.  It  is  particularly  prone  to  occur  in  the  sediment  after  the 
ingestion  of  certain  fruits  as  well  as  after  the  ingestion  of  benzoic  acid 
(see  page  300). 

Leucine  and  Tyrosine. — Leucine  and  tyrosine  have  frequently 
been  detected  in  the  urine,  either  in  solution  or  as  a  sediment.  Neither 
of  them  occurs  in  the  urine  ordinarily  ex- 
cept in  association  wuth  the  other,  /.  e., 
whenever  leucine  is  detected  it  is  more 
than  probable  that  tyrosine  accompanies 
it.  They  have  been  found  pathologi- 
cally in  the  urine  in  acute  yellow  atro- 
phy of  the  liver,  in  acute  phosphorus 
poisoning,  in  cirrhosis  of  the  liver,  in 
severe  cases  of  typhoid  fever  and  small-  ^ 

pox,  and  in  leukaemia.     In  urinary  sedi-       Fig.  ioq.— Crystals  of  Impure 

,        .  J-        -1  4.   u-  •  Leucine.     (Ogden.) 

ments  leucme  ordmaniy  crystalhzes  in 

characteristic  spherical  masses  which  show  both  radial  and  concentric 
striations  and  are  highly  refractive  (Fig.  109,  above).  Some  investi- 
gators claim  that  these  crystals  which  are  ordinarily  called  leucine  are, 
in  reality,  generally  urates.  This  view  point  has  become  more  general  in 
recent  years.  For  the  crystalline  form  of  pure  leucine  obtained  as  a 
decomposition  product  of  protein  see  Fig.  27.  p.  85.  T}Tosine  crystallizes 
in  urinary  sediments  in  the  well-known  sheaf  or  tuft  formation  (Fig. 
24,  p.  81).  For  other  tests  on  leucine  and  tyrosine  see  pages  ^90 
and  91. 

Haematoidin  and  Bilirubin. — There  are  divergent  opinions  regard- 
ing the  occurrence  of  these  bodies  in  urinary  sediment.     Each  of  them 


368  PHYSIOLOGICAL   CHEMISTRY. 

crystallizes  in  the  form  of  tufts  of  small  needles  or  in  the  form  of  small 
plates  which  are  ordinarily  yellowish-red  in  color  (Fig.  42,  p.  161).  Be- 
cause of  the  fact  that  the  crystalline  form  of  the  two  substances  is  identical 
many  investigators  claim  them  to  be  one  and  the  same  body.  Other 
investigators  claim,  that  while  the  crystalline  form  is  the  same  in  each 
case,  there  are  certain  chemical  differences  which  may  be  brought  out 
very  strikingly  by  properly  testing.  For  instance,  it  has  been  claimed 
that  haematoidin  may  be  differentiated  from  bilirubin  through  the  fact 
that  it  gives  a  momentary  color  reaction  (blue)  when  nitric  acid  is  brought 
in  contact  with  it,  and,  further,  that  it  is  not  dissolved  on  treatment  with 
ether  or  potassium  hydroxide.  Pathologically,  typical  crystals  of 
haematoidin  or  bilirubin  have  been  found  in  the  urinary  sediment 
in  jaundice,  acute  yellow  atrophy  of  the  liver,  carcinoma  of  the  liver, 
cirrhosis  of  the  liver,  and  in  phosphorus  poisoning,  typhoid  fever,  and 
scarlatina. 

Magnesium  Phosphate. — Magnesium  phosphate  crystals  occur 
rather  infrequently  in  the  sediment  of  urine  which  is  neutral, 
alkaline,  or  feebly  acid  in  reaction.  It  ordinarily  crystallizes  in  elon- 
gated, highly  refractive,  rhombic  plates  which  are  soluble  in  acetic 
acid. 

Indigo. — Indigo  crystals  are  frequently  found  in  urine  which  has 
undergone  alkaline  fermentation.  They  result  from  the  breaking  down 
of  indoxyl-sulphates  or  indoxyl-glycuronates.  Ordinarily  indigo  deposits 
as  dark  blue  stellate  needles  or  occurs  as  amorphous  particles  or  broken 
fragments.  These  crystalline  or  amorphous  forms  may  occur  in  the 
sediment  or  may  form  a  blue  film  on  the  surface  of  the  urine.  Indigo 
crystals  generally  occur  in  urine  which  is  alkaline  in  reaction,  but  they 
have  been  detected  in  acid  urine. 

Xanthine. — ^Xanthine  is  a  constituent  of  normal  urine  but  is  found 
in  the  sediment  in  crystalline  form  very  infrequently,  and  then  only  in 
pathological  urine.  When  present  in  the  sediment  xanthine  generally 
occurs  in  the  form  of  whetstone-shaped  crystals  somewhat  similar  in  form 
to  the  whetstone  variety  of  uric  acid  crystal.  They  may  be  differentiated 
from  uric  acid  by  the  great  ease  with  which  they  may  be  brought  into  solu- 
tion in  dilute  ammonia  and  on  applying  heat.  Xanthine  may  also  form 
urinary  calculi.  The  clinical  significance  of  xanthine  in  urinary  sediment 
is  not  well  understood. 

Melanin. — Melanin  is  an  extremely  rare  constituent  of  urinary 
sediments.  Ordinarily  in  melanuria  the  melanin  remains  in  solu- 
tion; if  it  separates  it  is  generally  held  in  suspension  as  fine  amorphous 
granules. 


URINE.  369 

(b)  Organized  Sediments. 

Epithelial  cells. 
Pus  cells. 

Hyaline. 

Granular. 

Epithelial. 
Casts.     I    Blood. 

Fatty. 

Waxy. 
I  Pus. 
Cylindroids. 
Erythrocytes. 
Spermatozoa. 
Urethral  filaments. 
Tissue  debris. 
Animal  parasites. 
Micro-organisms. 
Fibrin. 
Foreign  substances  due  to  contamination. 

Epithelial  Cells. — The  detection  of  a  certain  number  of  these  cells  in 
urinary  sediment  is  not,  of  itself,  a  pathological  sign,  since  they  occur  in 
normal  urine.  However,  in  certain  pathological  conditions  they  are 
greatly  increased  in  number,  and  since  different  areas  of  the  urinary  tract 
are  lined  with  different  forms  of  epithelial  cells,  it  becomes  necessary, 
when  examining  urinary  sediments,  to  note  not  only  the  relative  number 
of  such  cells,  but  at  the  same  time  to  carefully  observe  the  shape  of  the 
various  individuals  in  order  to  determine,  as  far  as  possible,  from  what 
portion  of  the  tract  they  have  been  derived.  Since  the  different  layers  of 
the  epithelial  lining  are  composed  of  cells  different  in  form  from  those  of 
the  associated  layers,  it  is  evident  that  a  careful  microscopical  examination 
of  these  cells  may  tell  us  the  particular  layer  which  is  being  desquamated. 
It  is  frequently  a  most  difficult  undertaking,  however,  to  make  a  clear 
differentiation  between  the  various  forms  of  epithelial  cells  present  in  the 
sediment.  If  skilfully  done,  such  a  miscropical  differentiation  may  prove 
to  be  of  very  great  diagnostic  aid. 

The  principal  forms  of  epithelial  cells  met  with  in  urinary  sediments 
are  shown  in  Fig.  no,  p.  370. 

Pus  Cells. — Pus  corpuscles  or  leucocytes  are  present  in  extremely 
small  numbers  in  normal  urine.  Any  considerable  increase  in  the  number, 
however,  ordinarily  denotes  a  pathological  condition,  generallv  an  acute 
24 


o/' 


PHYSIOLOGICAL    CHEMISTRY. 


or  chronic  inflammatory  condition  of  some  portion  of  the  urinary  tract. 
The  sudden  appearance  of  a  large  amount  of  pus  in  a  sediment  denotes 
the  opening  of  an  abscess  into  the  urinary  tract.  Other  form  elements, 
such  as  epithelial  cells,  casts,  etc.,  ordinarily  accompany  pus  corpuscles 
in  urinary  sediment  and  a  careful  examination  of  these  associated  elements 
is  necessary  in  order  to  form  a  correct  diagnosis  as  to  the  origin  of  the  pus. 
Protein  is  always  present  in  urine  which  contains  pus. 

The  appearance  which  pus  corpuscles  exhibit  under  the  microscope 
depends  greatly  upon  the  reaction  of  the  urine  containing  them.  In 
acid  urine  they  generally  present  the  appearance  of  round,  colorless  cells 


Fig.  no. — Epithelium  from  Different  Areas  of  the  Urinary  Tract. 
a,  Leucocyte  (for  comparison);  b,  renal  cells;  c,  superficial  pelvic  cells;  d,  deep  pelvic 
cells;  e,  cells  from  calices;/,  cells  from  ureter;  g,  g,  g,  g,  g,  squamous  epithelium  from  the 
bladder;  h,  h,  neck-of-bladder  cells;  i,  epithelium  from  prostatic  urethra;  k,  urethral  cells; 
/,  /,  scaly  epithelium;  m,  m',  cells  from  seminal  passages;  n,  compound  granule  cells;  o,  fatty 
renal  cell.     {Ogden.) 

composed  of  refractive,  granular  protoplasm,  and  may  frequently  exhibit 
amoeboid  movements,  especially  if  the  slide  containing  them  be  warmed 
slightly.  They  are  nucleated  (one  or  more  nuclei) ,  the  nuclei  being  clearly 
visible  only  upon  treating  the  cells  with  water,  acetic  acid,  or  some  other 
suitable  reagent.  In  urine  which  has  a  decided  alkaline  reaction,  on  the 
other  hand,  the  pus  corpuscles  are  often  greatly  degenerated.  They  may* 
be  seen  as  swollen,  transparent  cells,  which  exhibit  no  granular  structure 
and  as  the  process  of  degeneration  continues  the  cell  outline  ceases  to  be 
visible,  the  nuclei  fade,  and  finally  only  a  mass  of  debris  containing 
isolated  nuclei  and  an  occasional  cell  remains. 

It  is  frequently  rather  difficult  to  make  a  differentiation  between  pus 
corpuscles  and  certain  types  of  epithelial  cells  which  are  similar  in  form. 


URINE. 


371 


Such  confusion  may  be  avoided  by  the  addition  of  iodine  solution  (I  in 
KI),  a  reagent  which  stains  the  pus  corpuscles  a  deep  mahogany -brown 
and  transmits  to  the  epithelial  cells  a  light  yellow  tint.  The  test  proposed 
by  Vitali  often  gives  very  satisfactory  results.  This  simply  consists 
in  acidifying  the  urine  (if  alkaline)  with  acetic  acid,  then  filtering,  and 
treating  th^  sediment  on  the  filter  paper  with  freshly  prepared  tincture  of 
guaiac.  The  presence  of  i)us  in  the  sediment  is  indicated  if  a  blue  color 
is  observed.  Large  numbers  of  pus  corpuscles  are  present  in  the  urinary 
sediment  in  gonorrhoea,  leucorrhoea,  chronic  pyelitis,  and  in  abscess  of 


Fig.  m. — Pus  Corpuscles.     (After  Ultzmann.) 

I,  Normal;  2,  showing  amoeboid  movements;  3,  nuclei  rendered  distinct  by  acetic  acid;  4, 

as  observed  in  chronic  pyelitis;  5,  swollen  by  ammonium  carbonate. 

the  kidney.  In  addition  to  the  usual  constituents  found  in  leucocytes 
Mandel    and    Levene^  claim    that  pus  cells  contain  glucothionic  acid. 

Casts. — These  are  cylindrical  formations,  which  originate  in  the 
uriniferous  tubules  and  are  forced  out  by  the  pressure  of  the  urine.  They 
vary  greatly  in  size,  but  in  nearly  every  instance  they  possess  parallel 
sides  and  rounded  ends.  The  finding  of  casts  in  the  urine  is  very  impor- 
tant because  of  the  fact  that  they  generally  indicate  some  kidney  disorder; 
if  albumin  accompanies  the  casts  the  indication  is  much  accentuated. 
Casts  have  been  classified  according  to  their  microscopical  characteristics 
as  follows:  (a)  Hyaline,  {b)  granular,  (c)  epithelial,  {d)  blood,  (e)  fatty, 
ij)  waxy,  {g)  pus. 

(a)  Hyaline  Casts. — These  are  composed  of  a  basic  material  which 
is  transparent,  homogeneous,  and  very  light  in  color  (Fig.  112,  p.  372). 
In  fact,  chiefly  because  of  these  physical  properties,  they  are  the  most 

*  Mandel  and  Levene:   Biochemische  Zeilschri/t,  4,  78,  1907. 


372 


PHYSIOLOGICAL   CHEMISTRY, 


difficult  form  of  renal  casts  to  detect  under  the  microscope.  Frequently 
such  casts  are  impregnated  with  deposits  of  various  forms,  such  as  erythro- 
cytes, epithelial  cells,  fat  globules,  etc.,  thus  rendering  the  form  of  the  cast 
more  plainly  visible.  Staining  is  often  resorted  to  in  order  to  render  the 
shape  and  character  of  the  cast  more  easily  determined.  Ordinary  iodine 
solution  (I  in  KI)  may  be  used  in  this  connection;  many  of  the  aniline 
dyes  are  also  in  common  use  for  this  purpose,  e.  g.,  gentian-violet,  Bis- 
marck-brown, methylene-blue,  fuchsin,  and  eosin.  Generally,  but  not 
always,  albumin  is  present  in  urine  containing  hyaline  casts.     Hyaline 


Fig.  112. — Hyaline  Casts. 
One  cast  is  impregnated  with  four  renal  cells. 


casts  are  common  to  all  kidney  disorders,  but  occur  particularly  in  the 
earliest  and  recovering  stages  of  parenchymatous  nephritis  and  interstitial 
nephritis. 

{b)  Granular  Casts. — The  common  hyaline  material  is  ordinarily  the 
basic  substance  of  this  form  of  cast.  The  granular  material  generally 
consists  of  albumin,  epithelial  cells,  fat,  or  disintegrated  erythrocytes  or 
leucocytes,  the  character  of  the  cast  varying  according  to  the  nature  and 
size  of  the  granules  (Fig.  113,  p.  373,  and  Fig.  114,  page  374).  Thus 
we  have  casts  of  this  general  type  classified  a.s  finely  granular  and  coarsely 
granular  casts.  Granular  casts,  and  in  particular  the  finely  granular 
types,  occur  in  the  sediment  in  practically  every  kidney  disorder  but  are 


URINE. 


373 


probably    especially    characteristic    of    the    sediment    in  inflammatory 
disorders. 

(c)  Epithelial  Casts. — These  are  casts  bearing  upon  their  surface  epi- 
•  thelial  cells  from  the  lining  of  the  uriniferous  tubules  (Fig.  115,  p.  374). 
The  basic  material  of  this  form  of  cast  may  be  hyaline  or  granular  in 
nature.     Epithelial  casts  are  particularly  abundant  in  the  urinary  sedi- 
ment in  acute  nephritis. 

(b)  Blood  Casts. — Casts  of  this  type  may  consist  of  erythrocytes 
borne  upon  a  hyaline  or  a  fibrinous  basis  (Fig.  116,  p.  374).  The  occur- 
rence of  such  casts  in  the  urinary  sediment  denotes  renal  hemorrhage  and 
they  arc  considered  to  be  especially  characteristic  of  acute  diflfuse  nephritis 
and  acute  congestion  of  the  kidney. 


Fig.  113. — Granular  Casts.     {Mter  Peyer.) 

((?)  Fatty  Casts. — Fatty  casts  may  be  formed  by  the  deposition  of  fat 
globules  or  crystals  of  fatty  acid  upon  the  surface  of  a  hyaline  or  granular 
cast  (Fig.  117,  p.  375).  In  order  to  constitute  a  true  fatty  cast  the 
deposited  material  must  cover  the  greater  part  of  the  surface  area  of  the 
cast.  The  presence  of  fatty  casts  in  urinary  sediment  indicates  fatty 
degeneration  of  the  kidney;  such  casts  are  particularly  characteristic  of 
subacute  and  chronic  inflammation  of  the  kidney. 

(/)  Waxy  Casts. — These  casts  possess  a  basic  substance  similar  to 
that  which  enters  into  the  foundation  of  the  hyaline  form  of  cast.  In 
common  with  the  hyaline  type  they  are  colorless,  refractive  bodies,  but 
differ  from  this  form  of  cast  in  being,  in  general,  of  greater  length  and 


374 


PHYSIOLOGICAL    CHEMISTRY. 


diameter  and  possessing  sharper  outlines  and  a  light  yellow  color  (Fig 
ii8,  p.  375).  Such  casts  occur  in  several  forms  of  nephritis,  but  do 
not  appear  to  characterize  any  particular  type  of  the  disorder  except 
amyloid  disease,  in  which  they  are  rather  common. 


Fig.  114. — ^Granular  Casts. 
a,  Finely  granular;  b,  coarsely  granular. 


Fig.  115. — Epithelial  Casts. 


Fig.  116. — Blood,  Pus,  Hyaline  and  Epithelial  Casts. 
a,  Blood  casts;  b,  pus  cast;  c,  hyaline  cast  impregnated  with  renal  cells;  d,  epithelial  casts. 

(g)  Pus  Casts. — Casts  whose  surface  is  covered  with  pus  cells  or  leuco- 
cytes are  termed  pus  casts  (Fig.  116,  above).  They  are  frequently 
mistaken  for  epithelial  casts.  The  differentiation  between  these  two 
types  is  made  very  simple,  however,  by  treating  the  cast  with  acetic  acid 


URINE. 


375 


Fig.  117. — Fatty  Casts.     (After  Pcyer. 


Fig    iiS. — Fatty  and  Waxy  Casts. 
<j.  Fatty  casts;  b,  waxy  casts. 


376  PHYSIOLOGICAL   CHEMISTRY. 

which  causes  the  nuclei  of  the  leucocytes  to  become  plainly  visible.     The 
true  pus  cast  is  quite  rare  and  indicates  renal  suppuration. 

Cylindroids. — These  formations  may  occur  in  normal  or  pathological 
urine  and  have  no  particular  clinical  significance.  They  are  frequently 
mistaken  for  true  casts,  especially  the  hyaline  type,  but  they  are  ordinarily 
fiat  in  structure  with  a  rather  smaller  diameter  than  casts,  may  possess 
forked  or  branching  ends,  and  are  not  composed  of  homogenous  material 
as  are  the  hyaline  casts.  Such  "false  casts"  may  become  coated  with 
urates,  in  which  event  they  appear  granular  in  structure.     The  basic 


Fig.  119. — Cylindroids.     {Aiier:  Peyer.) 

substance  of  cylindroids  is  often  the  nucleoprotein  of  the  urine  (see  Fig. 
119,  above). 

Erythrocytes. — These  form  elements  are  present  in  the  urinary 
sediment  in  various  diseases.  They  appear  as  the  normal  biconcave, 
yellow  erythrocyte  (Plate  IV,  opposite  page  196)  or  may  exhibit  certain 
modifications  in  form,  such  as  the  crenated  type  (Fig.  120,  p.  377)  which 
is  often  seen  in  concentrated  urine.  Under  different  conditions  they  may 
become  swollen  sufficiently  to  entirely  erase  the  biconcave  appearance  and 
may  even  occur  in  the  form  of  colorless  spheres  having  a  smaller  diameter 
than  the  original  disc-shaped  corpuscles.  Erythrocytes  are  found  in 
urinary  sediment  in  hemorrhage  of  the  kidney  or  of  the  urinary  tract,  in 
traumatic  hemorrhage,  hemorrhage  from  congestion,  and  in  hemorrhagic 
diathesis. 

Spermatozoa. — Spermatozoa  may  be  detected  in  the  urinary  sedi- 


URINE. 


377 


ment  in  diseases  of  the  genital  organs,  as  well  as  after  coitus,  nocturnal 
emissions,  epileptic,  and  other  convulsive  attacks,  and  sometimes  in  severe 
febrile  disorders,  especially  in  typhoid  fever.     In  form  they  consist  of  an 


Fig.  I20. — Crexated  Erythrocytes. 


oval  body,  to  which  is  attached  a  long,  delicate  tail  (Fig.  121,  below). 
Upon  examination  they  may  show  motility  or  may  be  motionless. 

Urethral  Filaments. — These  are  peculiar  thread-like  bodies  which 


Fig.  121. — Human  Spermatozoa. 


are  sometimes  found  in  urinar}*  sediment.  They  may  occasionalh^  be 
detected  in  normal  urine  and  pathologically  are  found  in  the  sediment  in 
acute  and  chronic  gonorrhoea  and  in  urethrorrhoea.  The  ground-sub- 
stance of  these  urethral  filaments  is,  in  part  at  least,  similar  to  that  of  the 


378  PHYSIOLOGICAL    CHEMISTRY, 

cylindroids  (see  page  376).  The  urine  first  voided  in  the  morning  is  best 
adapted  for  the  examination  for  filaments.  These  filaments  may  ordi- 
narily be  removed  by  a  pipette  since  they  are  generally  macroscopic. 

Tissue  Debris. — Masses  of  cells  or  fragments  of  tissue  are  frequently 
found  in  the  urinary  sediment.  They  may  be  found  in  the  sediment  in 
tubercular  affections  of  the  kidney  and  urinary  tract  or  in  tumors  of  these 
organs.  Ordinarily  it  is  necessary  to  make  a  histological  examination  of 
such  tissue  fragments  before  coming  to  a  final  decision  as  to  their  origin. 

Animal  Parasites. — The  cysts,  booklets,  and  membrane  shreds 
of  echinococci  are  sometimes  found  in  the  urinary  sediments.  Other 
animal  organisms  which  are  more  rarely  met  with  in  the  urine  are  em- 
bryos of  the  Filaria  sanguinis  and  eggs  of  the  Distoma  hamatohium  and 
Ascarides.  Animal  parasites  in  general  occur  most  frequently  in  the 
urine  in  tropical  countries. 

Micro-organisms. — Bacteria  as  well  as  yeasts  and  moulds  are 
frequently  detected  in  the  urine.  Both  the  pathogenic  and  non-patho- 
genic forms  of  bacteria  may  occur.  The  non-pathogenic  forms  most 
frequently  observed  are  micrococcus  urece,  bacillus  urea,  and  staphylococcus 
urecB  liquefaciens .  Of  the  pathogenic  forms  many  have  been  observed, 
e.  g.,  Bacterium  Coli,  typhoid  bacillus,  tubercle  bacillus,  gonococcus,  bacillus 
pyocyaneus,  and  proteus  vulgaris.  Yeast  and  moulds  are  most  frequently 
met  in  diabetic  urine. 

Fibrin. — Following  haematuria,  fibrin  clots  are  occasionally  ob- 
served in  the  urinary  sediment.  They  are  generally  of  a  semi-gelatin- 
ous consistency  and  of  a  very  light  color,  and  when  examined  under 
the  microscope  they  are  seen  to  be  composed  of  bundles  of  highly  re- 
fractive fibers  which  run  parallel. 

Foreign  Substances  Due  to  Contamination. — Such  foreign  sub- 
stances as  fibers  of  silk,  linen,  or  wool;  starch  granules,  hair,  fat,  and 
sputum,  as  well  as  muscle  fibers,  vegetable  cells,  and  food  particles  are 
often  found  in  the  urine.  Care  should  be  taken  that  these  foreign 
substances  are  not  mistaken  for  any  of  the  true  sedimentary  constituents 
already  mentioned. 


CHAPTER  XXI. 
URINE :  CALCULI. 

Urinan'  calculi,  also  called  concrelians,  or  concrements  are  solid 
masses  of  urinary  sediment  formed  in  some  part  of  the  urinary  tract. 
They  vary  in  shape  and  size  according  to  their  location,  the  smaller 
calculi,  termed  sand  or  gravel,  in  general  arising  from  the  kidney  or  the 
pelvic  portion  of  the  kidney,  whereas  the  large  calculi  are  ordinarily 
formed  in  the  bladder.  There  are  two  general  classes  of  calculi  as 
regards  composition,  i.  e.,  simple  and  compound.  The  simple  form  is 
made  up  of  but  a  single  constituent,  whereas  the  compound  type  con- 
tains two  or  more  individual  constituents.  The  structural  plan  of 
most  calculi  consists  of  an  arrangement  of  concentric  rings  about  a 
central  nucleus,  the  number  of  rings  frequently  being  dependent  upon 
the  number  of  individual  constituents  which  enter  into  the  structure 
of  the  calculus.  In  case  two  or  more  calculi  unite  to  form  a  single  calculus 
the  resultant  body  will  obviously  contain  as  many  nuclei  as  there  were 
individual  calculi  concerned  in  its  construction.  Under  certain  condi- 
tions the  growth  of  a  calculus  will  be  principally  in  only  one  direction, 
thus  preventing  the  nucleus  from  maintaining  a  central  location.  The 
qualitative  composition  of  urinary  calculi  is  dependent,  in  great  part, 
upon  the  reaction  of  the  urine,  e.  g.,  if  the  reaction  of  the  urine  is  acid  the 
calculi  present  will  be  composed,  in  great  part  at  least,  of  substances  that 
are  capable  of  depositing  in  acid  urine. 

According  to  Ultzmann,  out  of  545  cases  of  urinary  calculus,  uric 
acid  and  urates  formed  the  nucleus  in  about  81  per  cent  of  the  cases; 
earthy  phosphates  in  about  9  per  cent;  calcium  oxalate  in  about  6  per 
cent;  cystine  in  something  over  i  per  cent,  while  in  about  3  per  cent 
of  the  cases  some  foreign  body  comprised  the  nucleus. 

In  the  chemical  examination  of  urinary  calculi  the  most  valuable 
data  are  obtained  by  subjecting  each  of  the  concentric  layers  of  the 
calculus  to  a  separate  analysis.  Material  for  examination  may  be 
conveniently  obtained  by  sawing  the  calculus  carefully  through  the 
nucleus,  then  separating  the  various  layers  or  by  scraping  off  from 
each  layer  (without  separating  the  layers)  enough  powder  to  conduct 
the  examination  as  outlined  in  the  scheme  (see  page  381). 

379 


380  PHYSIOLOGICAL   CHEMISTRY. 

Varieties  of  Calculus. 

Uric  Acid  and  Urate  Calculi. — Uric  acid  and  urates  constitute  the 
nuclei  of  a  large  proportion  (81  per  cent)  of  urinary  concretions.  Such 
stones  are  always  colored,  the  tint  varying  from  a  pale  yellow  to  a 
brownish-red.  The  surface  of  such  calculi  is  generally  smooth  but  it 
may  be  rough  and  uneven. 

Phosphatic  Calculi. — Ordinarily  these  concretions  consist  prin- 
cipally of  "triple  phosphate"  and  other  phosphates  of  the  alkaline 
earths,  with  very  frequent  admixtures  of  urates  and  oxalates.  The 
surface  of  such  calculi  is  generally  rough  but  may  occasionally  be  rather 
smooth.  The  calculi  are  somewhat  variable  in  color,  exhibiting  gray, 
white,  or  yellow  tints  under  different  conditions.  When  composed  of 
earthy  phosphates  the  calculi  are  characterized  by  their  friability. 

Calcium  Oxalate  Calculi.^ — This  is  the  hardest  form  of  calculus 
to  deal  with,  and  is  rather  difficult  to  crush.  They  ordinarily  occur 
in  two  general  forms,  i.  e.,  the  small,  smooth  concretion  which  is  charac- 
terized as  the  hemp-seed  calculus  and  the  medium-sized  or  large  stone 
possessing  an  extremely  uneven  surface  which  is  generally  classed  as 
a  mulberry  calculus.  This  roughened  surface  of  the  latter  form  of  calcu- 
lus is  due,  in  many  instances,  to  protruding  calcium  oxalate  crystals  of 
the  octahedral  type. 

Calcium  Carbonate  Calculi.— Calcium  carbonate  concretions  are 
quite  common  in  herbivorous  animals,  but  of  exceedingly  rare  occurrence 
in  man.  They  are  generally  small,  white,  or  grayish  calculi,  spherical 
in  form  and  possess  a  hard,  smooth  surface. 

Cystine  Calculi. — The  cystine  calculus  is  a  rare  variety  of  calculus. 
Ordinarily  they  occur  as  small,  smooth,  oval,  or  cylindrical  concretions 
which  are  white  or  yellow  in  color  and  of  a  rather  soft  consistency. 

Xanthine  Calculi. — This  form  of  calculus  is  somewhat  more  rare 
than  the  cystine  type.  The  color  may  vary  from  white  to  brownish- 
yellow.  Very  often  uric  acid  and  urates  are  associated  with  xanthine 
in  this  type  of  calculus.  Upon  rubbing  a  xanthine  calculus  it  has  the 
property  of  assuming  a  wax-like  appearance. 

Urostealith  Calculi. — This  form  of  calculus  is  extremely  rare. 
Such  concretions  are  composed  principally  of  fat  and  fatty  acid.  When 
moist  they  are  soft  and  elastic,  but  when  dried  they  become  brittle. 
Urostealiths  are  generally  light,  in  color. 

Fibrin  Calculi. — Fibrin  calculi  are  produced  in  the  process  of 
blood  coagulation  within  the  urinary  tract.  They  frequently  occur  as 
nuclei  of  other  forms  of  calculus.     They  are  rarely  found. 


URINE. 


381 


On  Heating  the  Powder  on  Platinum  Foil,  li 


Does  not  burn 


Does  burn 


The  powder  when  treated  with  HCl 
Does  not  effervesce 


The  powder  gently  heated  with  HCl 


The  fX)wder  when  moist- 
ened with  a  little  KOH 


3  < 


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382  PHYSIOLOGICAL    CHEMISTRY. 

Cholesterol  Calculi. — An  extremely  rare  form  of  calculus  some- 
what resembling  the  cystine  type. 

Indigo  Calculi. — Indigo  calculi  are  extremely  rare,  only  two  cases 
having  been  reported.  One  of  these  indigo  calculi  is  on  exhibition  in 
the  museum  of  Jefferson  Medical  College  of  Philadelphia. 

The  scheme,  proposed  by  Heller  and  given  on  page  381,  will  be  found 
of   much   assistance  in   the   chemical   examination   of  urinary   calculi. 


CHAPTER  XXII. 
URINE :  QUANTITATIVE  ANALYSIS. 

I.  Protein. 

1.  Scherer's  Coagulation  Method. — The  content  of  coagulablc 
protein  may  be  accurately  determined  as  follows:  Place  50  c.c.  of  urine  in 
a  small  beaker  and  raise  the  temperature  of  the  fluid  to  about  40°  C.  upon 
a  water-bath.  Add  dilute  acetic  acid,  drop  by  drop,  to  the  warm  urine, 
to  precipitate  the  protein  which  will  separate  in  a  flocculent  form.  Care 
should  be  taken  not  to  add  too  much  acid;  ordinarily  less  than  twenty 
drops  is  sufficient.  The  temperature  of  the  water  in  the  water-bath 
should  now  be  raised  to  the  boiling-point  and  maintained  there  for  a  few 
minutes  in  order  to  insure  the  complete  coagulation  of  the  protein  present. 
Now  filter  the  urine ^  through  a  previously  washed,  dried,  and  weighed 
filter  paper,  wash  the  precipitated  protein,  in  turn,  with  hot  water,  95 
per  cent  alcohol,  and  with  ether,  and  dry  the  paper  and  precipitate,  to 
constant  weight,  in  an  air-bath  at  110°  C.  Subtract  the  weight  of  the 
filter  paper  from  the  combined  weight  of  the  paper  and  precipitate  and 
calculate  the  percentage  of  protein  in  the  urine  specimen. 

Calculation. — To  determine  the  percentage  of  protein  present  in  the 
urine  under  examination,  multiply  the  weight  of  the  precipitate,  expressed 
in  grams,  by  2. 

2.  Esbach's  Method. — This  method  depends  upon  the  precipitation 
of  protein  by  Esbach's  reagent"  and  the  apparatus  used  in  the  estimation  is 
Esbach's  albuminometer  (Fig.  122,  p.  384).  In  making  a  determin- 
ation fill  the  albuminometer  to  the  point  U  with  urine,  then  intro- 
duce the  reagent  until  the  point  R  is  reached.  Now  stopper  the 
tube,  invert  it  slowly  several  times  in  order  to  insure  the  thorough  mixing 
of  the  fluids,  and  stand  the  tube  aside  for  24  hours.  Creatinine,  resin 
acids,  etc.,  are  precipitated  in  this  method,  and  for  this  and  other  reasons 
it  is  not  as  accurate  as  the  coagulation  method.  It  is,  however,  extensively 
used  clinically.     According  to  Sahli^  the  method  is  "accurate  approxi- 

'  If  it  is  desired  the  precipitate  may  be  filtered  off  on  an  imweighed  paper,  and  its  nitrogen 
content  determined  by  the  Kjeldahl  method  (see  p.  401).  In  order  to  arrive  at  correct  figures 
for  the  protein  content  it  is  then  simply  necessary  to  multiply  the  total  nitrogen  content  by 
6.25  (see  p.  438).  Correction  should  be  made  for  the  nitrogen  content  of  the  filter  paper 
used  unless  this  factor  is  negligible. 

^  Esbach's  reagent  is  prepared  by  dissolving  10  grams  of  picric  acid  and  20  grams  of 
citric  acid  in  i  liter  of  water. 

^Sahli:  Lehrbuch  d.  klin.  Untersuchungs-Methoden,  5th  Aufl.,  1909. 

383 


384 


PHYSIOLOGICAL    CHEMISTRY. 


lili 


mately  to  one  part  per  1000,"  whereas  Pfeiffer^  claims  it  is  not  accurate 
for  less  than  one-half  or  for  more  than  five  parts  per  1000. 

Calculatimi. — The  graduations  on  the  albuminometer  indicate  grams 
of  protein  per  liter  of  urine.  Thus,  if  the  protein  precipitate  is  level  with 
the  figure  3  of  the  graduated  scale,  this  denotes  that  the  urine  examined 
contains  3  grams  of  protein  to  the  liter.  To  express  the 
amount  of  protein  in  per  cent  simply  move  the  decimal 
point  one  place  to  the  left.  In  the  case  under  con- 
sideration the  urine  contains  0.3  per  cent  protein. 

3.  Kwilecki's  Modification  of  Esbach's  Method.^ 
— Add  10  drops  of  a  10  per  cent  solution  of  FeClg  to  the 
acid  urine  before  introducing  the  Esbach's  reagent. 
Warm  the  tube  and  contents  in  a  water-bath  at  72°  C. 
for  5-6  minutes  and  make  the  reading. 

II.  Dextrose. 

I.  Fehling's  Method. — Place  10  c.c.  of  the  urine 
under  examination  in  a  100  c.c.  volumetric  flask  and 
make  the  volume  up  to  100  c.c.  with  distilled  water. 
Thoroughly  mix  this  diluted  urine  by  pouring  it  into  a 
beaker  and  stirring  with  a  glass  rod,  then  transfer  a 
portion  of  it  to  a  burette  which  is  properly  supported  in 
a  clamp. 

Now  place  10  c.c.  of  Fehling's  solution-''  in  a  small 
beaker,  dilute  it  with  approximately  40  c.c.  of  distilled 
water,  heat  to  boiling,  and  observe  whether  decomposi- 
tion of  the  Fehling's  solution  itself  has  occurred  as 
indicated  by  the  production  of  a  turbidity.  If  such  tur- 
bidity is  produced  the  Fehling's  solution  is  unfit  for  use. 
Clamp  the  burette  containing  the  dilute  urine  immedi- 
ately over  the  beaker  and  carefully  allow  from  0.5  to  i 
c.c.  of  the  diluted  urine  to  flow  into  the  boiling  Fehl- 
ing's solution.  Bring  the  solution  to  the  boiling-point 
after  each  addition  of  urine  and  continue  running  the  urine  from  the 
burette,  0.5-1  c.c.  at  a  time,  as  indicated,  until  the  Fehling's  solution  is 
completely  reduced,  i.  e.,  until  all  the  cupric  oxide  in  solution  has  been 
precipitated  as  cuprous  oxide.  This  point  will  be  indicated  by  the 
absolute  disappearance  of  all  blue  color.     When  this  end-point  is  reached 

'Pfeiffer:  Berl.  klin.  Woch.,  49,  114,  1912. 
'  Kwilecki:    Miittch.  Med.  Woch.,  56,  p.  1330. 

'  Directions  for  the  i)reparalion  of  Fehling's  solution  are  given  in  a  note  at  the  bottom 
of  page  32. 


Fig.  122. — Es- 
bach's Albumin- 
ometer.   (Ogden.) 


urine:  quantitative  analysis.  385 

note  the  number  of  cubic  centimeters  of  diluted  urine  used  in  the  pro- 
cess and  calculate  the  percentage  of  dextrose  present,  in  the  sample  of 
urine  analyzed,  according  to  the  method  given  below. 

This  is  a  very  satisfactory  method,  the  main  objection  to  its  use  being 
the  uncertainty  attending  the  determination  of  the  end-reaction,  i.  e.,  the 
difficulty  with  which  the  exact  point  where  the  blue  co\or finally  disappears 
is  noted.  Several  means  of  accurately  fixing  this  point  have  been  sug- 
gested, but  they  are  practically  all  open  to  objection.  As  good  a  "check" 
as  any,  perhaps  is  to  filter  a  few  drops  of  the  solution,  through  a  double 
paper,  after  the  blue  color  has  apparently  disappeared,  acidify  the  filtrate 
with  acetic  acid  and  add  potassium  ferrocyanide.  If  the  copper  of  the 
Fehling's  solution  has  been  completely  reduced,  there  will  be  no  color 
reaction,  whereas  the  production  of  a  brown  color  indicates  the  presence 
of  unreduced  copper.  Harrison  has  recently  suggested  the  following  pro- 
cedure to  determine  the  e.xact  end-point:  To  about  i  c.c.  of  a  starch 
iodide  solution^  in  a  test-tube  add  2-3  drops  of  acetic  acid  and  introduce 
into  the  acidified  mixture  1-2  drops  of  the  solution  to  be  tested. 
Unreduced  copper  will  be  indicated  by  the  production  of  a  purplish-red  or 
blue  color  due  to  the  liberation  of  iodine. 

It  is  ordinarily  customary  to  make  at  least  three  determinations  by 
Fehling's  method  before  coming  to  a  final  conclusion  regarding  the  sugar 
content  of  the  urine  under  examination. 

Calculation. — Ten  c.c.  of  Fehling's  solution  is  completely  reduced  by 
0.05  gram  of  dextrose.  ^  If  y  represents  the  number  of  cubic  centimeters  of 
undiluted  urine  (obtained  by  dividing  the  burette  reading  by  10)  necessary 
to  reduce  the  10  c.c.  of  Fehling's  solution,  we  have  the  following  proportion: 

y  :  0.05  ::  100  :  x  (percentage  of  dextrose). 

2.  Benedict's  Method  No.  i. — To  30  c.c.  of  Benedict's  solution*  in 
a  small  beaker  add  from  2.5  grams  to  5  grams  of  anhydrous  sodium  car- 

*  The  starch-iodide  solution  may  be  prepared  as  follows:  Mix  o.i  gram  of  starch  with 
cold  water  in  a  mortar  and  pour  the  suspended  starch  granules  into  75-100  c.c.  of  boiling 
water,  stirring  continuously.  Cool  the  starch  paste,  add  20-25  grams  of  potassium  iodide 
and  dilute  the  mixture  to  250  c.c.  This  solution  deteriorates  upon  standing,  and  therefore 
must  be  freshly  prepared  as  needed. 

-  The  values  for  certain  other  sugars  are  as  follows: 

Lactose 0.0676  gram. 

Maltose o .  074    gram. 

Invert  sugar 0.0475  gram. 

'  Benedict's  solution  used  in  the  quantitative  determination  of  sugar  consists  of  three 
separate  solutions.  The  copper  sulphate  solution  and  the  alkaline  tartrate  solution  are  the  same 
as  those  already  described  in  connection  with  Benedict's  qualitative  test,  see  p.  ^^.  The 
third  solution  is  made  up  as  follows: 

Potassium  ferro-thiocyanate  soluticm  =  i$  grams  of  potassium  ferrocyanide,  62.5  grams 
of  potassium  thiocyanate  and  50  grams  of  anhydrous  sodium  carbonate  dissolved  in  water 
and  made  up  to  500  c.c. 

These  three  solutions  should  be  preserved  separately  in  rubber-stoppered  bottles  and 
mixed  in  equal  volumes  when  needed  for  use.     This  is  done  to  prevent  deterioration. 

25 


386  PHYSIOLOGICAL    CHEMISTRY. 

bonate^  and  heat  the  mixture  to  boihng  over  a  wire  gauze  until  the  car- 
bonate has  been  brought  into  solution. 

Place  the  urine  under  examination  in  a  burette  and  run  it  into  the  hot 
Benedict  solution  rather  rapidly^  until  the  formation  of  a  heavy  chalk- 
white  precipitate  is  noted  and  the  blue  color  of  the  solution  lessens  per- 
ceptibly in  its  intensity.  From  this  point  in  the  determination  from  2  to 
10  drops^  of  the  urine  should  be  run  into  the  boiling  Benedict  solution  at 
one  time,  boiling  the  solution  vigorously  for  about  15  seconds  after  each 
addition.  Complete  reduction  of  the  copper  is  indicated  here  as  in 
Fehling's  original  method,  by  the  complete  disappearance  of  all  blue  color. 
The  end-point  here,  however,  is  very  sharply  defined,  contrary  to  the 
conditions  in  the  older  method. 

To  prevent  the  annoying  bumping  which  often  interferes  with  the 
titration,  a  medium-sized  piece  of  washed  absorbent  cotton*  may  be 
introduced  into  the  solution.  This  cotton  may  be  stirred  about  through 
the  solution  as  the  titration  proceeds  and  the  bumping  thus  eliminated. 

Calculation. — Thirty  cubic  centimeters  of  Benedict's  solution  is 
completely  reduced  by  0.073  gram  of  dextrose.  If  y  represents  the  num- 
ber of  cubic  centimeters  of  urine  necessary  to  reduce  the  30  c.c.  of  the 
solution  we  have  the  following  proportion: 

y  :  0.073  ••  i°°  •  ^  (percentage  of  dextrose). 

Benedict's  Method  No.  2.^ — "The  urine,  10  c.c.  of  which  should  be 
diluted  with  water  to  100  c.c.  (unless  the  sugar  content  is  believed  to  be 
low),  is  poured  into  a  50  c.c.  burette  up  to  the  zero  mark.  Twenty-five 
cubic  centimeters  of  the  reagent"  are  measured  with  a  pipette  into  a  por- 
celain evaporation  dish  (25-30  cm.  in  diameter),  10  to  20  grams  of 
crystallized  sodium  carbonate  (or  one-half  the  weight  of  the  anhydrous 

'  The  amount  added  depends  upon  the  dilution  to  which  the  solution  is  to  be  subjected 
in  titration.  For  this  reason  the  maximum  amount  of  sodium  carbonate  should  be  added 
when  titrating  urines  containing  a  ver}'  low  percentage  of  sugar. 

^  Not  rapidly  enough,  however,  to  interfere  in  any  marked  degree  with  the  continuous 
vigorous  })oiling  of  the  solution. 

^  The  e.xact  amount  to  run  in  depends  upon  the  intensity  of  the  remaining  blue  color, 
as  well  as  upon  the  sugar  content  of  the  urine.  The  lo  drops  should  be  added  at  one  time 
only  when  urines  containing  a  very  low  percentage  of  sugar  are  under  examination. 

*  Glass  wool  may  be  substituted  if  desired. 

*  Benedict:  Jour.  Am.  Med.  Ass'n.,  57,  1193,  191 1. 

"         Copper  sulphate  (crystallized) 18 .0  grams. 

Sodium  carbonate    (cn>-stallixed,  one-half    the    weight  of  the 

anhydrous  salt  may  be  used) 200 .0  grams. 

Sodium  or  potassium  citrate 200.0  grams. 

Potassium  thiocyanate 125 .0  grams. 

Potassium  ferrocyanide  (5  per  cent  solution) 5.0  c.c. 

Distilled  water  to  make  a  total  volume  of 1000. o  c.c. 

With  the  aid  of  heat  dissolve  the  carbonate,  citrate  and  thiocyanate  in  enough  water  to 
make  aViOut  800  c.c.  of  the  mixture  and  filter  if  necessary.  Dissolve  the  copper  sulphate 
separately  in  about  100  c.c.  of  water  antl  pour  the  solution  slowly  into  the  other  licjuid,  with 
constant  stirring.  Add  the  ferrocyanide  solution,  cool  and  dilute  to  exactly  i  liter.  Of  the 
various  constituents,  the  copper  salt  only  need  be  weighed  with  exactness.  Twenty-five 
cubic  centimeters  of  the  reagent  are  reduced  by  50  mg.  of  glucose. 


urine:  quantitative  analysis.  387 

salt)  arc  added,  together  with  a  small  (juantity  of  powdered  pumice  stone 

or  talcum,  and  the  mixture  heated  to  boiling  over  a  free  flame  until  the 

carbonate  has  entirely  dissolved.     The  diluted  urine  is  now  run  in  from 

the  burette,  rather  rapidly  until  a  chalk-white  precipitate  forms,  and  the 

blue  color  of  the  mixture  begins  to  lessen  perceptibly,  after  which  the 

solution  from  the  burette  must  be  run  in  a  few  drops  at  a  time,  until  the 

disappearance  of  the  last  trace  of  blue  color,  which  marks  the  end-point. 

The   solution   must  be   kept   vigorously   boiling  throughout   the   entire 

titration.     If  the  mixture  becomes  too  concentrated  during  the  process, 

water  may  be  added  from  time  to  time  to  replace  the  volume  lost  by 

evaporation.     The  calculation  of  the  percentage  of  sugar  in  the  original 

sample  of  urine  is  very  simple.     The  25  c.c.   of  copper  solution  are 

reduced  by  exactly  50  mg.  of  glucose.     Therefore  the  volume  run  out  of 

the  burette  to  effect  the  reduction  contained  50  mg.  of  the  sugar.     When 

the  urine  is  diluted  1:10,  as  in  the  usual  titration  of  diabetic  urines,  the 

formula  for  calculating  the  per  cent  of  the  sugar  is  the  following: 

o.oso  .         .  .     ,  ,         ,        .      -^   .       ,  , 

^    Xiooo=per  cent  m   origmal  sample,  wherem   X  is   the   number 

of  cubic  centimeters  of  the  diluted  urine  required  to  reduce  25  c.c. 
of  the  copper  solution." 

"In  the  use  of  this  method  chloroform  must  not  be  present  during  the 
titration.  If  used  as  a  preservative  in  the  urine  it  may  be  removed  by 
boiling  a  sample  for  a  few  minutes,  and  then  diluting  to  its  original 
volume." 

"Like  the  reagent  for  qualitative  employment,  the  one  for  quantitative 
work  will  keep  indefinitely  after  its  preparation.  As  regards  the  accuracy 
of  the  method,  it  may  be  stated  that  repeated  determinations,  and  com- 
parisons with  results  by  the  polariscope  and  by  Allihn's  gravimetric 
process  have  shown  the  method  to  be  probably  more  exact  than  any  other 
titration  method  available  for  sugar  work." 

3.  Purdy's  Method. — Purdy's  solution^  is  a  modification  of  Fehling's 
solution  and  is  said  to  possess  greater  stability  than  the  latter.  One  of 
the  most  satisfactory  points  about  the  method  as  suggested  by  Purdy  is 
the  ease  with  which  the  exact  end-reaction  may  be  determined.  In 
determining  the  percentage  of  dextrose  by  this  method  proceed  as  follows: 

'  Purdy's  solution  has  the  following  composition: 

Copper  sulphate 4-752  grams. 

Potassium  hydroxide 23  .5      grams. 

Ammonia  (U.  S.  P.,  sp.  gr.  0.9) 35°  o      c.c. 

Glycerol 38.0      c.c. 

Distilled  water,  to  make  total  volume  i  liter. 
In  preparing  the  solution  bring  the  copper  sulphate  and  potassium  hydroxide  into  solution 
in  separate  vessels,  mix  the  two  solutions,  cool  the  mixture,  and  add  the  ammonia  and  glycerol. 
After  this  has  been  done  the  total  volume  should  be  made  up  to  i  liter  with  distilled  water. 

Thirty-five  cubic  centimeters  of  Purdy's  solution  is  exactly  reduced  by  0.02  gram  of 
dextrose. 


388  PHYSIOLOGICAL   CHEMISTRY. 

Place  35  c.c.  of  Purdy's  solution  in  a  200  c.c.  Erlenmeyer  flask  and  dilute 
the  fluid  with  approximately  two  volumes  of  distilled  water.  Fit  a  cork, 
provided  with  two  perforations,  to  the  neck  of  the  flask  and  through  one 
perforation  introduce  the  tip  of  a  burette  and  through  the  second  perforation 
introduce  a  tube  bent  at  right  angles  in  such  a  manner  as  to  allow  the  steam 
to  escape  and  keep  the  fumes  of  ammonia  away  from  the  face  of  the  oper- 
ator as  completely  as  possible.^  Now  bring  the  solution  to  the  boiling- 
point  and  add  the  urine,  drop  by  drop,  untfl  the  intensity  of  the  blue  color 
begins  to  diminish.  When  this  point  is  reached  add  the  urine  somewhat 
more  slowly  until  the  blue  color  is  entirely  dissipated  and  an  absolutely 
decolorized  solution  remains.  Take  the  burette  reading  and  calculate 
the  percentage  of  dextrose  in  the  urine  examined  according  to  the  method 
given  below. 

Care  should  be  taken  not  to  boil  the  solution  for  too  long  a  period, 
since,  under  these  conditions,  sufficient  ammonia  might  be  lost  to  allow 
the  cuprous  hydroxide  to  precipitate. 

Some  investigators  consider  it  to  be  advisable  to  dilute  the  urine 
before  applying  the  above  manipulation,  but  ordinarily  this  is  not  neces- 
sary unless  the  urine  has  a  high  content  of  dextrose  (5  per  cent  or  over). 
In  this  event  the  urine  may  be  diluted  with  2-3  volumes  of  water  and 
the  proper  correction  made  in  the  calculation. 

Calculation. — -Thirty-five  c.c.  of  Purdy's  solution  is  completely  reduced 
by  0.02  gram  of  dextrose.  If  y  represents  the  number  of  cubic  centimeters 
of  undiluted  urine  necessary  to  reduce  35  c.c.  of  Purdy's  solution,  we 
have  the  following  proportion: 

y  :  0.02  : :  100  :  x  (percentage  of  dextrose). 

4.  Fermentation  Method. — This  method  consists  in  the  measure- 
ment of  the  volume  of  carbon  dioxide  evolved  when  the  dextrose  of  the 
urine  undergoes  fermentation  with  yeast.  None  of  the  various  methods 
whose  manipulation  is  based  upon  this  principle  is  absolutely  accurate. 
The  method  in  which  Einhorn's  saccharometer  (Fig.  3,  page  36)  is  the 
apparatus  employed  is  perhaps  as  satisfactory  as  any  for  clinical  pur- 
poses. The  procedure  is  as  follows:  Place  about  15  c.c.  of  urine  in  a 
mortar,  add  about  i  gram  of  yeast  (1/16  of  the  ordinary  cake  of  com- 
pressed yeast)  and  carefully  crush  the  latter  by  means  of  a  pestle.  Trans- 
fer the  mixture  to  the  saccharometer,  being  careful  to  note  that  the 
graduated  tube  is  completely  filled  and  that  no  air  bubbles  gather  at  the 

*  This  side  tube  may  also  be  equipped  with  a  simple  air-valve,  thus  insuring  the  exclusion 
of  aiir  and  thereby  contributing  to  the  accuracy  of  the  determination,  inasmuch  as  the  cuprous 
salts  would  be  reoxidized  upon  coming  in  contact  with  the  air.  If  one  is  careful  to  maintain 
iJie  solution  continuously  at  the  boiling-point  throughout  the  entire  process,  however,  there 
is  no  opportunity  for  air  to  enter  and  therefore  no  need  of  an  air-valve. 


urine:  quantitative  analysis.  389 

top.  Allow  the  apparatus  to  stand  in  a  warm  place  (30°  C.)  for  12  hours 
and  observe  the  percentage  of  dextrose  as  indicated  by  the  graduated 
scale  of  the  instrument.  Both  the  percentage  of  dextrose  and  the  number 
of  cubic  centimeters  of  carbon  dioxide  are  indicated  by  the  graduations 
on  the  side  of  the  saccharometer  tube. 

The  availability  of  the  fermentation  procedure  as  a  quantitative  aid 
has  been  appreciably  lowered  through  the  important  findings  of  Neuberg 
and  Associates^  recently  reported.  They  show  that  yeast  has  the  prop- 
erty of  splitting  off  carbon  dioxide  from  the  carboxyl  group  of  amino  and 
other  aliphatic  acids.  The  active  agent  in  this  "sugar-free  fermentation" 
is  an  enzyme  called  carboxylase.  Inasmuch  as  amino  acids  are  always 
present  in  the  urine,  the  error  from  this  source  is  apparent. 

5.  Polariscopic  Examination.— Before  subjecting  urine  to  a 
polariscopic  examination  the  slightly  acid  fluid  should  be  decolorized 
as  thoroughly  as  possible  by  the  addition  of  a  little  lead  acetate.  The 
urine  should  be  well  stirred  and  then  filtered  through  a  filter  paper  which 
has  not  been  previously  moistened.  In  this  way  a  perfectly  clear  and 
almost  colorless  liquid  is  obtained. 

In  determining  dextrose  by  means  of  the  polariscope  it  should  be 
borne  in  mind  that  this  carbohydrate  is  often  accompanied  by  other 
optically  active  substances,  such  as  proteins,  laevulose,  /9-oxybutyric 
acid,  and  conjugate  glycuronates  which  may  introduce  an  error  into 
the  polariscopic  reading;  the  method  is,  however,  sufficiently  accurate 
for  practical  purposes. 

For  directions  as  to  the  manipulation  of  the  polariscope  see  page  36. 

III.  Uric  Acid. 

I.  Folin-Shaffer  Method. — Introduce  100  ex.-  of  urine  into  an 
Erlenmeyer  flask,  add  25  c.c.  of  the  Folin-Shaffer  reagent*  and  after 
shaking  the  flask  to  thoroughly  mix  the  fluids  allow  the  mixture  to 
stand,'  with  or  without  further  stirring,  until  the  precipitate  has  settled 
(5-10  minutes).  Filter,  transfer  100  c.c.  of  the  filtrate  to  a  200  c.c. 
Erlenmeyer  flask,  add  5  c.c.  of  concentrated  ammonium  hydroxide 
and  allow  the  mixture  to  stand  for  24  hours.  Transfer  the  precipitated 
ammonium  urate  quantitatively  to  a  filter  paper,"  using   10  per  cent 

*  Neuberg  and  Associates:    Biochemische  Zeitschrift,  31,  170;  36  (60,  68,  and  76),  191 1. 

^  It  is  preferable  to  use  more  than  100  c.c.  of  urine  if  the  fluid  has  a  specific  gravity  less 
than  1.020. 

^  The  Folin-Shaffer  reagent  consists  of  500  grams  of  ammonium  sulphate,  5  grams  of 
uranium  acetate  and  60  c.c.  of  10  per  cent  acetic  acid  in  650  c.c.  of  distilled  water. 

*  The  mi.\ture  should  not  be  allowed  to  stand  for  too  long  a  time  at  this  point,  since  uric 
acid  may  be  lost  through  precipitation. 

*  The  Schleicher  and  Schiill  hardened  papers  or  the  Baker  and  Adamson  washed,  ashless 
variety  are  ver}'  satisfactory'  for  this  purpose. 


390  PHYSIOLOGICAL    CHEMISTRY. 

ammonium  sulphate  to  remove  the  final  traces  of  the  urate  from  the 
flask.  Wash  the  precipitate  approximately  free  from  chlorides  by 
means  of  lo  per  cent  ammonium  sulphate  solution/  remove  the  paper 
from  the  funnel,  open  it,  and  by  means  of  hot  water  rinse  the  precipitate 
back  through  the  funnel  into  the  flask  in  which  the  urate  was  originally 
precipitated.  The  volume  of  fluid  at  this  point  should  be  about  loo  c.c. 
Cool  the  solution  to  room  temperature,  add  15  c.c.  of  concentrated 
sulphuric  acid  and  titrate  at  once  with  N/20  potassium  permanganate, 
K^Mn^O^,  solution.  The  first  tinge  of  pink  color  which  extends  through- 
out the  fluid  after  the  addition  of  two  drops  of  the  permanganate  solution, 
while  stirring  with  a  glass  rod,  should  be  taken  as  the  end-reaction. 
Take  the  burette  reading  and  compute  the  percentage  of  uric  acid  present 
in  the  urine  under  examination. 

Calculation. — Each  cubic  centimeter  of  N/20  potassium  permanga- 
nate solution  is  equivalent  to  3.75  milligrams  (0.00375  gram)  of  uric 
acid.  The  100  c.c.  from  which  the  ammonium  urate  was  precipitated 
is  equivalent  to  only  four-fifths  of  the  100  c.c.  of  urine  originally  taken, 
therefore  we  must  take  five-fourths  of  the  burette  reading  in  order  to 
ascertain  the  number  of  cubic  centimeters  of  the  permanganate  solution 
required  to  titrate  100  c.c.  of  the  original  urine  to  the  correct  end-point. 
If  y  represents  the  number  of  cubic  centimeters  of  the  permanganate 
solution  required,  we  may  make  the  following  calculation: 

y  X  0.00375  =  weight  of  uric  acid  in  100  c.c.  of  urine. 

Because  of  the  solubility  of  the  ammonium  urate  a  correction  of  3 
milligrams  should  be  added  to  the  final  result. 

Calculate  the  quantity  of  uric  acid  in  the  twenty-four-hour  urine 
specimen. 

2.  Heintz  Method. — This  is  a  very  simple  method  and  was  the 
first  one  in  general  use  for  the  quantitative  determination  of  uric  acid. 
It  is  believed  to  be  somewhat  less  accurate  than  the  method  just  described. 
The  procedure  is  as  follows:  Place  100  c.c.  of  filtered  urine  in  a  beaker, 
add  5  c.c.  of  concentrated  hydrochloric  acid,  stir  the  fluid  thoroughly, 
and  stand  it  away  in  a  cool  place  for  24  hours.  Filter  off  the  uric  acid 
crystals  upon  a  washed,  dried  and  weighed  filter  paper  and  wash  them 
with  cold  distilled  water,  a  few  cubic  centimeters  at  a  time  until  the 
chlorides  are  removed.  Now  wash,  in  turn,  with  alcohol  and  with 
ether  and  finally  dry  the  paper  and  crystals  to  constant  weight  at  110°  C. 
In  the  process  of  washing  the  uric  acid  free  from  chlorides  an  error  is 
introduced,  since  every  cubic  centimeter  of  water  so  used  dissolves 
0.00004  gram  of  uric  acid.^     For  this  reason  a  correction  is  necessary. 

'  This  washing  may  be  conveniently  done  l^y  decantalion  if  desired,  thus  retaining  the 
major  portion  of  the  precipitate  in  the  flask. 

^  His  and  Paul:    Zeit.  physiol.  Chem.,  31,1,  1900. 


URINF. :    QUANTITATIVE    ANALYSIS.  39I 

It  has  been  suggested  that  the  pigment  of  the  crystals  is  equivalent  in 
weight  to  the  amount  of  uric  acid  dissolved  by  the  first  30  c.c.  of  water, 
and  this  factor  should  be  taken  into  account  in  the  computation  of  the 
percentage  of  uric  acid. 

Calculation. — Since  100  c.c.  of  urine  was  used  the  corrected  weight 
of  the  uric  acid  crystals,  in  grams,  will  express  the  percentage  of  uric  acid 
present. 

3.  Kriiger  and  Schmidt's  Method. — This  method  serves  for  the 
detection  of  both  uric  acid  and  the  purine  bases.  The  principle  in- 
volved is  the  precipitation  of  both  the  uric  acid  and  the  purine  bases 
in  combination  with  copper  oxide  and  the  subsequent  decomposition 
of  this  precipitate  by  means  of  sodium  sulphide.  The  uric  acid  is  then 
precipitated  by  means  of  hydrochloric  acid  and  the  purine  bases  are 
separated  from  the  filtrate  in  the  form  of  their  copper  or  silver  com- 
pounds. The  nitrogen  content  of  the  precipitates  of  uric  acid  and  pur- 
ine bases  is  then  determined  by  means  of  the  Kjeldahl  method  (see  p. 
401)  and  the  corresponding  values  for  uric  acid  and  purine  bases  calcu- 
lated. The  method  is  as  follows:  To  400  c.c.  of  albumin-free  urine ^ 
in  a  liter  flask,"  add  24  grams  of  sodium  acetate,  40  c.c.  of  a  solution 
of  sodium  bisulphite'  and  heat  the  mixture  to  boiling.  Add  40-80 
c.c*  of  a  ID  per  cent  solution  of  copper  sulphate  and  maintain  the  tem- 
perature of  the  mixture  at  the  boiling-point  for  at  least  three  minutes. 
Filter  off  the  flocculent  precipitate,  wash  it  with  hot  water  until  the 
wash  water  is  colorless,  and  return  the  washed  precipitate  to  the  flask 
by  puncturing  the  tip  of  the  filter  paper  and  washing  the  precipitate 
through  by  means  of  hot  water.  Add  water  until  the  volume  in  the 
flask  is  appro.ximately  200  c.c,  heat  the  mixture  to  boiling,  and  decom- 
pose the  precipitate  of  copper  oxide  by  the  addition  of  30  c.c.  of  sodium 
sulphide  solution.^  After  decomposition  is  complete,  the  mixture  should 
be  acidified  with  acetic  acid  and  heated  to  boiling  until  the  separating 
sulphur  collects  in  a  mass.  Filter  the  hot  fluid  by  means  of  a  filter  pump, 
wash  with  hot  water,  add  10  c.c.  of  10  per  cent  hydrochloric  acid  and 
evaporate  the  filtrate  in  a  porcelain  dish  until  the  total  volume  has  been 
reduced  to  about  ten  cubic  centimeters.     Permit  this  residue  to  stand 

'  If  albumin  is  present,  the  urine  should  be  heated  to  boiling,  acidified  with  acetic  acid 
and  filtered. 

-  The  total  volume  of  urine  for  the  twenty-four  hours  should  be  suflSciently  diluted  with 
water  to  make  the  total  volume  of  the  solution  1600-2000  c.c. 

'  .\  solution  containing  50  grams  of  Kahlbaum's  commercial  sodium  bisulphite  in  100 
A.  of  water. 

*  The  e.xact  amount  depending  upon  the  content  of  the  purine  bases. 

•*  This  is  made  by  saturating  a  i  per  cent  solution  of  soduim  hydro.xide  with  hydrogen 
sulphide  gas  and  adding  an  equal  volume  of  i  per  cent  sodium  hydroxide. 

Ordinarily  the  addition  of  30  c.c.  of  this  solution  is  sufficient,  but  the  presence  of  an  excess 
of  sulphide  should  be  prtrcen  by  adding  a  drop  of  lead  acetate  to  a  drop  of  the  solution.  Under 
these  conditions  a  dark  brown  color  will  show  the  presence  of  an  excess  of  sodium  sulphide. 


592 


PHYSIOLOGICAL    CHEMISTRY. 


about  two  hours  to  allow  for  the  separation  of  the  uric  acid,  leaving  the 
purine  bases  in  solution.  Filter  off  the  precipitate  of  uric  acid,  using  a 
small  filter  paper,  and  wash  the  uric  acid,  with  water  made  acid  with 
sulphuric  acid,  until  the  total  volume  of  the  original  filtrate  and  the  wash 
water  aggregates  75  c.c.  Determine  the  nitrogen  content  of  the  precipi- 
tate by  means  of  the  Kjeldahl  method  (see  p.  401)  and  calculate  the  uric 
acid  equivalent. 

Calculation. — In  calculating  the  uric  acid  value  from  the  total  nitrogen 
simply  multiply  the  latter  by  three  and  add  0.0035  to  the  product  as  a 
correction  for  the  uric  acid  remaining  in  solution  in  the  75  c.c. 

IV.  Urea. 


I.  Knop-Hiifner  Hypobromite  Method  (using  Marshall's  Urea 
Apparatus). — Place  the  thumb  over  the  side  opening  of  the  bulbed- 

tube  of  the  apparatus  (Fig.  123)  and  care- 
fully fill  the  tube  with  sodium  hypobromite 
solution. '  Close  the  opening  in  the  end  of 
the  tube  with  a  rubber  stopper,  incline  the 
tube  to  allow  air-bubbles  to  escape,  and 
finally  invert  the  tube  and  fix  the  stoppered 
end  in  the  saucer-shaped  vessel.  By  means 
of  the  graduated  pipette  rapidly  introduce 
I  c.c.  of  urine^  into  the  hypobromite  solu- 
tion through  the  side  opening  of  the  bulbed- 
tube.  Withdraw  the  pipette  immediately 
after  the  urine  has  been  introduced.  When 
the  decomposition  of  the  urea  is  completed 
(10-20  minutes)  gently  tap  the  bulbed-tube 
with  the  finger  in  order  to  dislodge  any  gas 
bubbles  which  may  have  collected  on  the 
inner  surface  of  the  glass.     The  atmospheric 

Fig.  123.— Marshall's  Urea      pressure  should  now  be  equalized  by  attach- 
Apparatus.     (Tyson.)  .  i     n      i       -l  i 

a,  Bulbed  measuring  tube;  b,  mg  the  funnel-tube  to  the  bulbcd-tubc  at  the 
^peTitt'funnd-tube?  '"'"'''  ^^de  opening  and  introducing  hypobromite 

solution  into  it  until  the  columns  of  liquid  in 

'  The  ingredients  of  tlie  sodium  hypobromite  solution  should  be  prepared  in  the  form 
of  two  separate  solutions.  When  needed  for  use  mix  one  volume  of  solution  a,  one  volume 
of  solution  b,  and  3  volumes  of  water. 

(a)  Dissolve  125  grams  of  sodium  bromide  in  water,  add  125  grams  of  bromine  and 
make  the  total  volume  of  the  solution  i  liter. 

(b)  A  solution  of  sodium  hydroxide  having  a  specific  gravity  of  1.250.  This  is  approxi- 
mately a  22.5  per  cent  solution. 

Preserve  both  solutions  in  rubber-stoppered  bottles. 

^  Ordinarily  i  c.c.  of  urine  is  sufficient;  more  may  be  used,  however,  if  its  content  of 
urea  is  very  low. 


'hiimmiiiiiiiniiM'i 


urine:  quantitative  analysis.  393 

the  two  tubes  are  uniform  in  hcij^ht.  The  graduated  scale  of  the  bulbed- 
tube  should  now  be  read  in  order  to  determine  the  number  of  cubic 
centimeters  of  nitrogen  gas  evolved.  By  means  of  the  appended  formula 
the  weight  of  the  urea  present  in  the  urine  under  examination  may  be 
computed. 

Calculatiou.^ — By  properly  substituting  in  the  following  formula 
the  weight  of  urea,  in  grams,  contained  in  the  volume  of  urine  decom- 
posed (i  c.c.  or  more)  may  readily  be  determined: 


w 


vip-T) 


354.5  X  760(1  +  0.003665/) 
w;  =  weight  of  urea,  in  grams. 

1*  =  observed  volume  of  nitrogen  expressed  in  cubic  centimeters. 
/>  =  barometric  pressure  expressed  in  mm.  of  mercury. 
T  =  tension  of  aqueous  vapor^  for  temperature  /. 
/  =  temperature  (centigrade). 

If  we  wish  to  calculate  the  percentage  of  urea  we  may  do  so  by  means  of 
the  following  proportion  in  which  y  represents  the  volume  of  urine  used 
and  w  denotes  the  weight  of  the  urea  contained  in  the  volume  y: 

y  :w  ::x:  (percentage  of  urea) . 

Sodium  hypobromite  solution  may  also  be  employed  for  the  deter- 
mination of  urea  in  the  apparatus  devised  by  Hiifner  which  is  pictured  in 
Fig.  124,  page  394. 

2.  Knop-Hiifner  Hypobromite  Method  (Using  the  Doremus- 
Hinds  Ureometer). — In  common  wdth  the  method  already  described, 
this  method  depends  upon  the  measurement  of  the  volume  of  nitrogen 
gas  liberated  when  the  urea  of  the  urine  is  decomposed  by  means  of  sodium 
hypobromite  solution.  The  Doremus-Hinds  ureometer  (Fig.  125,  p.  395), 
is  one  of  the  simplest  and  cheapest  forms  of  apparatus  in  general  use  for 
the  determination  of  urea  by  the  hypobromite  process.  In  using  this 
apparatus  proceed  as  follows:  Fill  the  side  tube  B  and  the  lumen  of  the 
stopcock  C  with  the  urine  under  examination.  Carefully  wash  out  tube 
A  with  water  and  introduce  into  it  sodium  hypobromite  solution,^  being 

*  0.003665=  coefficient  of  expansion  of  gases  for  1°  C.     354.5  =  number  of  c.c.  of  nitrogen 
gas  evolved  from  i  gram  of  urea. 

*  The  values  of  T  for  the  temperatures  ordinarily  met  with  are  given  in  the  following 
table: 

Temp.                       Tension  in  mm.                   Temp.                       Tension  in  mm. 
15°  C 12.677  21°  C 18.505 


16°  C 13 

17°  C 14 

18°  C 15 

19°  C 16 

20°  C. 17 


519  22°  C 19675 

009  23°  C 20.909 

351  24°  C 22.211 

345  25°  C 23.582 

396 


'For  directions  as  to  the  preparation  of  this  solution  see  page  392. 


394 


PHYSIOLOGICAL    CHEMISTRY. 


careful  to  fill  the  bulb  sufficiently  full  to  prevent  the  entrance  of  air  into 
the  graduated  portion.  Now  allow  i  c.c.  of  urine  ^  to  flow  from  tube  B  into 
tube  A  and  after  the  evolution  of  gas  bubbles  has  ceased  (10-20  minutes) 
take  the  reading  of  the  graduated  scale  on  tube  A. 

In  common  with  all  other  methods  which  are  based  upon  the  decom- 
position of  urea  by  means  of  hypobro- 
mite  solution,  this  method  is  not  abso- 
lutely correct.  It  is,  however,  suffi- 
ciently accurate  for  ordinary  clinical 
purposes. 

Calculation. — Observe  the  reading 
on  the  graduated  scale  of  tube  A.  This 
tube  is  so  graduated  as  to  represent  the 
weight  of  urea,  in  grams,  per  cubic 
centimeter  of  urine.  If  we  wish  to  com- 
pute the  percentage  of  urea  present  this 
may  be  done  very  readily  by  simply 
moving  the  decimal  point  two  places  to 
the  right;  e.  g.,  if  the  reading  is  0.02 
gram  the  urine  contains  2  per  cent  of 
urea. 

3.  Folin's  Method. — This  is  one  of 
the  most  accurate  methods  yet  devised 
for  the  determination  of  urea  in  the 
urine.  It  has,  however,  been  replaced 
to  a  great  extent  by  the  very  recent 
modification  of  Folin  and  Pettibone  (see 
p.  397).  The  procedure  is  as  follows: 
Place  5  c.c.  of  urine  in  a  200  c.c.  Erlen- 
meyer  flask  and  add  to  it  5  c.c.  of  con- 
centrated hydrochloric  acid,  20  grams  of 
crystallized  magnesium  chloride,  a  piece 
of  paraffin  the  size  of  a  hazel  nut,  and 
2-3  drops  of  a  I  per  cent  aqueous  solution  of  "alizarin  red."  Insert  a 
Folin  safety  tube  (Fig.  126,  p.  396)  into  the  neck  of  the  flask  and  boil  the 
mixture  until  each  drop  of  reflow  from  the  safety  tube  produces  a  very 
perceptible  bump;  the  heat  is  then  reduced  somewhat  and  continued  one 
and  one-half  hours.  The  contents  of  the  flask  must  not  remain  alkaline, 
and  to  obviate  this,  at  the  first  appearance  of  a  reddish  tinge  in  the  con- 
tents of  the  flask  a  few  drops  of  the  acid  distillate  are  shaken  back  into 

'  If  the  content  of  urea  in  the  urine  under  examination  is  large,  the  urine  may  be  diluted 
with  water  before  determining  the  urea.  If  this  is  done  it  must  of  course  be  taken  into  con- 
sideration in  ( ompuling  the  content  of  urea. 


Fig.  124. — Hufner's  Urea  Apparatus. 


urine:  quantitative  analysis. 


395 


r^ 


the  flask.  At  the  end  of  i  1/2  hours  the  contents  of  the  vessel  are 
transferred  to  a  i-liter  flask  with  about  700  c.c.  of  distilled  water, 
about  20  c.c.  of  10  per  cent  potassium  hydroxide  or  sodium  hydroxide 
solution  is  added  and  the  mixture  distilled  into  a  known  volume  of  N/io 
sulphuric  acid  until  the  contents  of  the  flask  are  nearly  dry  or  until  the 
distillate  fails  to  give  an  alkaline  reaction  to  litmus,  showing  the  absence 
of  ammonia.  The  time  devoted  to  this 
])rocess  is  ordinarily  about  an  hour.  Boil 
the  distillate  a  few  moments  to  free  it  from 
CO,,  then  cool  and  titrate  the  mixture  with 
X/io  sodium  hydroxide,  using  "alizarin 
red"  as  indicator. 

A  "check"  experiment  should  always 
be  made  to  determine  the  original  am- 
monia content  of  the  urine  and  of  the 
magnesium  chloride,  if  it  is  not  absolutely 
pure,  which  of  course  should  be  subtracted 
from  the  total  amount  of  ammonia  as  deter- 
mined by  the  above  process. 

The  Folin  method  is  extremely  accu- 
rate under  all  conditions  except  when  the 
urine  contains  sugar.  When  this  is  the 
case  the  carbohydrate  and  the  urea  unite, 
upon  being  heated,  and  form  a  very  stable 
combination.  For  this  reason  the  Folin 
method  is  not  suitable  for  use  in  the  exam- 
ination of  such  urines.  Under  such  condi- 
tions the  combination  Morner-Sjoqvist- 
Folin  method  which  is  given  below  or  the 
ipethod  of  Folin  and  Denis  (p.  398)  may 
be  used. 

4.  Morner-Sjoqvist-Folin  Method. — As  has  already  been  stated  in 
the  last  experiment,  this  method  excels  the  Folin  method  in  accuracy  only 
in  the  determination  of  urea  in  the  presence  of  carbohydrate  bodies. 
Briefly,  the  procedure  is  as  follows:^  Bring  the  major  portion  of  1.5  gram 
of  powdered  barium  hydroxide  into  solution  in  5  c.c.  of  urine  in  a  small 
flask,  and  treat  the  mixture  with  100  c.c.  of  an  alcohol-ether  solution, 
consisting  of  two  volumes  of  97  per  cent  alcohol  and  one  volume  of  ether. 
Stopper  the  flask  and  allow  it  to  stand  12-24  hours.  Filter  off  the  pre- 
cipitate, wash  it  with  the  alcohol-ether  mixture  and  remove  the  alcohol 

'  The  original  description  of  the  method  may  be  found  in  an  article  by  Morner:  Skatj- 
dinavisches  Archiv  Jilr  Physiologic,  14,  21)7,  1903. 


Fig.  125. — DoREMus-IIixDS 
Ureometer. 


396 


PHYSIOLOGICAL    CHEMISTRY. 


Q^=£J!fe 


and  ether  from  the  filtrate  by  distillation,  being  careful  to  keep  the  tempera- 
ture of  the  mixture  below  50°  C.  ^  Treat  the  remaining  fluid  (about  25  c.c.) 
with  2  c.c.  of  hydrochloric  acid  (sp.  gr.  1.124),  transfer  it  carefully  to  a 
200  c.c.  flask,  and  evaporate  the  mixture  to  dryness  on  a  water-bath. 
Now  add  20  grams  of  crystallized  magnesium  chloride  and  2  c.c.  of  con- 
centrated hydrochloric  acid  to  the  residue,  and  after  fitting  the  flask  with 

a  return  cooler  boil  the  mixture  on  a  wire 
gauze  over  a  small  flame  for  two  hours. 
Cool  the  solution,  dilute  to  750  c.c.  or  1000 
c.c.  with  water,  render  the  mixture  alkaline 
with  potassium  hydroxide  or  sodium  hy- 
droxide, distil  off  the  ammonia  and  collect 
it  in  an  acid  solution  of  known  strength. 
Boil  the  distillate  to  remove  carbon  dioxide, 
cool  and  titrate  with  an  alkali  of  known 
strength.  In  this  method,  as  well  as  in 
Folin's  method  (see  p.  394),  correction 
must  be  made  for  the  ammonia  originally 
present  in  the  urine  and  in  the  magnesium 
chloride. 

5.  Benedict's  Method.^ — Five  cubic 
centimeters  of  urine  are  introduced  into  a 
rather  wide  Jena  glass  test-tube,  about  3 
grams  of  potassium  bisulphate  and  1-2 
grams  of  zinc  sulphate^  added,  a  small 
quantity  of  powdered  pumice  and  a  bit  of 
paraffin  are  introduced  and  the  mixture 
boiled  almost  to  dryness  either  over  a  free 
flame  or  by  immersion  in  a  sulphuric  acid 
bath  at  about  130°.  The  tubes  are  then 
weighted  (a  screw  clamp  is  convenient)  and 
immersed  for  three-fourths  of  their  length  in  a  bath  of  sulphuric  acid 
at  a  temperature  of  162-165°  i^^t  lower)  for  one  hour. 

The  contents  of  the  tube  are  then  washed  into  an  800  c.c.  Kjeldahl 
distillation  flask,  diluted  to  about  400  c.c.  with  water,  made  alkaline 
by  the  addition  of  15-20  c.c.  of  10  per  cent  KOH  (or  25  c.c.  15  per  cent 
Na2C03)  and  distilled  as  usual  in  the  Kjeldahl  method  (page  401).  The 
value  obtained  must  be  corrected  for  ammonia. 

'  There  is  some  decomposition  of  urea  at  60°  C. 
^  Benedict:    Jour.  Biol.  Chem.,  8,  405,  igii. 

^  An  excess  of  zinc  salt  is  to  be  avoided  as  too  large  quantity  tends  to  cause  slight  frothing 
during  the  final  distillation. 


Fig..  126. — Folin's  Urea 
Apparatus. 


urine:  quantitative  analysis.  397 

Wclker*  has  suggested  an  electrical  bath  for  use  in  the  first  part  of  this 
method. 

6.  Method  of  Folin  and  Pettibone,  No.  i.^ — By  means  of  an  Ostwald 
pipette  (see  page  403)  introduce  i  c.c.  of  urine  into  a  Jena  test-tube 
(20-25  mm.  by  200  mm.).  Add  three  good-sized  drops  of  pure  phos- 
phoric acid,  one  drop  of  indicator  (alizarin)  and  a  few  grains  of  talcum 
powder  and  concentrate  the  mixture  to  one-half  its  volume  by  boiling 
over  a  free  flamC  for  2-3  minutes.  At  the  end  of  this  time  heat  the  test- 
tube  in  a  bath  of  sulphuric  acid,  oil,  or  parafhn,  for  fifteen  minutes  at 
a  temperature  of  175-180°  C.^  By  this  means  the  urea  is  decomposed 
with  the  formation  of  ammonium  phosphate.  Dissolve  the  contents  of 
the  tube  in  water  (1-2  c.c.)  with  the  aid  of  heat,  make  alkahne  with 
potassium  hydroxide*  (0.5-1  c.c.  of  a  50  per  cent  solution)  and  remove 
the  liberated  ammonia  by  means  of  a  strong  air  current  (see  page  404). 
This  process  requires  approximately  ten  minutes.  The  ammonia  may 
be  collected  in  25  c.c.  of  Ar/50  hydrochloric  acid  and  the  excess  acid 
titrated  with  N/ioo  sodium  hydroxide  using  alizarin  as  indicator. 

In  calculating  the  urea  value  a  correction  must  be  made  for  the 
ammonia  content  of  the  urine. 

With  the  bath  previously  heated  to  the  proper  temperature  the  above 
method  may  be  completed  in  about  one-half  hour. 

7.  Method  of  Folin  and  Pettibone,  No.  2.^— Dilute  the  urine  so 
that  I  c.c.  contains  0.75-1.5  mg.  of  urea  nitrogen.  Generally  dilutions 
of  1:20  or  1:10,  depending  on  the  concentration,  are  satisfactory.  By 
means  of  an  Ostwald  pipette  (see  page  403)  introduce  i  c.c.  of  the 
diluted  urine  into  a  large  dry  Jena  test-tube  (20-25  "^"i.  by  200  mm.) 
which  already  contains  7  grams  of  dry  ammania-free  potassium  acetate' 
{free  from  lumps),  i  c.c.  of  50  per  cent  acetic  acid,  a  small  sand  pebble 
or  a  little  powdered  zinc  (not  zinc  dust)  to  prevent  bumping  during  boiling, 
and  a  temperature  indicator.'^ 

'  Wclker:   Biochemical  Bulletin,  i,  439.  1912. 
^  Folin  and  Pettibone:  Jour.  Biol.  Chem.,  11,  512,  1912. 

^  The  bath  should  be  at  this  temperature  when  the  tubes  are  introduced.  Welker's  elec- 
tric bath  may  be  used  in  this  connection.     (See  Biochemical  Bulletin,  i,  439,  1912). 

*  Potassium  hydroxide  is  preferred  to  sodium  hydroxide  because  of  the  greater  solubility 
of  potassium  phosphate. 

*  Folin  and  Pettibone:  Jour.  Biol.  Chem.,  11,  513,  1912. 

*  A  satisfactory  preparation  containing  less  than  i  per  cent  of  moisture  and  free  from 
ammonia  may  be  obtained  from  J.  T.  Baker  Chemical  Co.,  Phillipsburg,  N.  J. 

^  "  This  temperature  indicator  consists  of  powdered  chloride-iodide  of  mercury  (HglCl) 
inclosed  in  a  sealed  glass  bulb  not  over  i  mm.  in  diameter.  This  salt  is  bright  red  at  ordinary 
temperatures.  At  118°  C.  it  turns  yellow  and  melts  to  a  clear  dark  red  liquid  at  155°  C. 
It  solidifies  again  at  about  148°  C  and  resumes  its  red  color  gradually  only  in  the  course  of 
about  twenty-four  hours.  The  melting-point  temperature,  153°  C,  is  fortunately  a  tempera- 
ture very  readily  obtained  and  maintained  by  means  of  potassium  acetate  and  as  the  acetate 
begins  to  cake  and  solidify  at  160-161°  C,  there  is  no  danger  in  this  combination  of  having 
either  too  high  or  too  low  a  temperature  without  its  being  unmistakably  apparent. 

The  HglCl  may  be  prepared  by  heating,  in  a  drj-  state,  intimately  mixed  mercuric  chloride 


398  PHYSIOLOGICAL   CHEMISTRY. 

Close  the  test-tube  by  means  of  a  rubber  stopper  carrying  an  empty 
narrow  "calcium  chloride  tube  "(i-5  cm.  by  25  cm.,  without  bulb)  as  a 
condenser.  Suspend  the  test-tube  and  condenser  above  a  micro-burner 
(see  page  403)  by  means  of  a  burette  clamp  or  some  similar  device  in  such 
a  way  that  they  may  be  easily  raised  or  lowered.  Heat  gently,  using  a 
bottomless  beaker  or  some  similar  device  as  a  wind  shield  if  needed.  The 
acetate  will  soon  dissolve  (two  minutes)  and  the  mixture  begin  to  boil. 
At  this  point  the  indicator  begins  to  melt  showing  that  the  desired  tem- 
perature (153-160°  C.)  has  been  reached.  Continue  the  boiling  in  a 
gentle,  even  manner  for  ten  minutes  at  the  end  of  which  time  the  decom- 
position of  the  urea  is  complete.  Remove  the  apparatus  from  the  flame 
and  dilute  the  contents  with  5  c.c.  of  water.  ^  Add  an  excess  of  alkali 
(2  c.c.  of  a  saturated  solution  of  sodium  hydroxide  or  potassium  carbonate) 
and  remove  the  liberated  ammonia  by  means  of  a  strong  air  current  (see 
page  404).  The  ammonia  may  be  caught  in  a  100  c.c.  volumetric  flask 
which  contains  about  35  c.c.  of  ammonia-free  water  and  2  c.c.  of  N/10 
acid.  With  a  strong  air  current  this  process  requires  only  about  ten 
minutes.  Determine  the  ammonia  colorimetrically  against  i  mg.  of 
nitrogen  in  the  form  of  ammonium  sulphate.  For  the  colorimetric 
procedure  see  the  total  nitrogen  determination,  page  402. 

8.  Method  of  Folin  and  Denis. ^ — Sugar  interferes  with  the  decompo- 
sition of  urea.  This  was  formerly  believed  to  be  due  to  the  formation  of 
nitrogenous  "melanins,"^  but  is  more  probably  due  to  the  formation  of 
definite,  stable  ureids."*  This  difficulty  may  be  overcome  by  proper  dilu- 
tion of  the  urine  thus  preventing  the  formation  of  the  ureids.  Because 
of  this  great  dilution  the  usual  titration  procedures  are  inappHcable,  and 
the  following  colorimetric  procedure  is  suggested: 

Dilute  I  c.c.  of  the  urine  with  20  to  100  volumes  of  ammonia-free 
water  and  decompose  i  c.c.  of  this  dilute  urine  with  potassium  acetate 
and  acetic  acid  as  described  under  the  method  of  Folin  and  Pettibone, 
No.  2,  on  page  397. 

By  means  of  an  air  current  remove  the  ammonia  to  a  second  test-tube 
which  contains  about  2  c.c.  of  water  and  0.5  c.c.  of  N/io  hydrochloric 
acid.  Add  to  the  contents  of  this  tube  about  2  c.c.  of  water  and  3  c.c.  of 
the  diluted  (i :  5)  Nessler- Winkler  solution  (page  404).     Wash  this  colored 

and  mercuric  iodide  in  molecular  proportions  at  150-160°  C.  for  6-8  hours.  At  the  end  of  the 
heating  the  product  should  be  powdered  and  used  as  it  is  for  it  cannot  be  puriiied  by  the  use 
of  solvents.  It  should  be  kept  dry  until  sealed  up  as  indicated."  These  temperature  indi- 
cators may  be  obtained  ready  prepared  in  tubes  from  Eimer  &  yVmend,  New  York. 

*  This  water  should  be  added  by  means  of  a  [ji[)ette  through  the  calcium  chloride  tube  so  as 
to  rinse  the  sides  of  the  tube  and  the  bottom  of  the  rubber  stof)per  from  any  possible  traces  of 
ammonium  acetate.     Not  more  than  5  c.c.  of  water  should  be  used  for  this  purpose. 

^  Folin  and  Denis;  Jour.  Biol.  Chem.,  11,  520,  igi2. 
'  Momer:  Skand.  Arch.  Physiol.,  14,  319. 

*  Folin:  Am.  Jour.  Physiol.,  13,  46,  1905. 


urine:  quantitative  analysis.  399 

solution  into  a  lo  c.c.  volumetric  flask  and  dilute  it  to  the  mark  with 
ammonia-free  water.  Transfer  the  entire  volume  to  a  dry  cylinder  of  a 
Duboscq  colorimeter  and  determine  the  dej)th  of  color  against  a  standard 
containing  i  mg.  of  nitrogen  per  loo  c.c.  of  solution.  For  the  detailed 
colorimetric  procedure  see  the  method  for  total  nitrogen,  page  402. 

V.  Ammonia. 

I.  Folin's  Method. — Place  25  c.c.  of  urine  in  an  aerometer  cylinder, 
30-40  cm.  in  height  (Fig.  127,  below),  add  about  i  gram  of  dry  sodium 
carbonate  and  introduce  some  crude  petroleum  to  prevent  foaming. 
Insert  into  the  neck  of  the  cylinder  a  rubber  stopper  provided  with  two 


Fig.  127. — FoLixs  .-ViiMoxiA  Apparatus. 

perforations,  into  each  of  which  passes  a  glass  tube,  one  of  which  reaches 
below  the  surface  of  the  liquid.  The  shorter  tube  (10  cm.  in  length) 
is  connected  with  a  calcium  chloride  tube  filled  with  cotton,  and  this  tube 
is  in  turn  joined  to  a  glass  tube  extending  to  the  bottom  of  a  500  c.c.  wide- 
mouthed  flask  which  is  intended  to  absorb  the  ammonia  and  for  this  pur- 
pose should  contain  20  c.c.  of  N  10  sulphuric  acid,  200  c.c.  of  ammonia- 
free  distilled  water  and  a  few  drops  of  an  indicator  (alizarin  red  or  congo 
red).  To  insure  the  complete  absorption  of  the  ammonia  the  absorption 
flask  is  provided  with  a  Folin  improved  absorption  tube  (Fig.  128,  p.  400) 
which  is  very  effective  in  causing  the  air  passing  from  the  cylinder  to 
come  into  intimate  contact  with  the  acid  in  the  absorption  flask.  In 
order  to  exclude  any  error  due  to  the  presence  of  ammonia  in  the  air  a  simi- 
lar absorption  apparatus  to  the  one  just  described  is  attached  to  the  other 
side  of  the  aerometer  cylinder,  thus  insuring  the  passage  of  ammonia-free 


400 


PHYSIOLOGICAL    CHEMISTRY. 


Fig.  1 2  8. — Folin 
Improved  Absorp- 
tion Tube. 


air  into  the  cylinder.  With  an  ordinary  filter  pump  and  good  water  pres- 
sure the  last  trace  of  ammonia  should  be  removed  from  the  cylinder  in 
about  one  and  one-half  hours.  ^  The  number  of  cubic 
centimeters  of  the  N/io  sulphuric  acid  neutralized  by 
the  ammonia  of  the  urine  may  be  determined  by  di- 
rect titration  with  N/io  sodium  hydroxide. 

This  is  one  of  the  most  satisfactory  methods  yet 
devised  for  the  determination  of  ammonia.  Steele^ 
has  recently  suggested  a  modification  for  use  on 
urines  containing  triple  phosphate  sediments.  In  this 
modification  .05-1.0  of  NaOH  and  about  15  grams 
of  NaCl  are  substituted  for  the  NagCOg  of  the  Folin 
method. 

Calculation. — Subtract  the  number  of  cubic  centi- 
meters of  N/io  sodium  hydroxide  used  in  the  titra- 
tion from  the  number  of  cubic  centimeters  of  N/io 
sulphuric  9,cid  taken.  The  remainder  is  the  number 
of  cubic  centimeters  of  N/io  sulphuric  acid  neutralized 
by  the  NH^  of  the  urine,  i  c.c.  of  N/io  sulphuric 
acid  is  equivalent  to  0.0017  ^^«^^^  of  NH^.  Therefore 
if  y  represents  the  volume  of  urine  used  in  the  deter- 
mination and  y'  the  number  of  cubic  centimeters  of  N/io  sulphuric  acid 
neutralized  by  the  NH^  of  the  urine,  we  have  the  following  proportion : 

y  :  100  :  :y'  X0.0017  :  x  (percentage  of  NH3  in  the  urine  examined). 

Calculate  the  quantity  of  NHg  in  the  twenty-four-hour  urine  specimen. 

2.  Method  of  Folin  and  Macallum.'' — By  means  of  Ostwald  pipettes 
(page  403)  introduce  1-5  c.c.  of  urine"*  into  a  Jena  test-tube  (20-25  mm, 
by  200  mm.)  and  add  to  the  urine  a  few  drops  of  a  solution  containing 
10  per  cent  of  potassium  carbonate  and  15  per  cent  of  potassium  oxalate. 
To  prevent  foaming  add  a  few  drops  of  kerosene  or  heavy,  crude  machine 
oil.  Pass  a  strong  air  current  (see  page  404)  through  the  mixture  until 
the  ammonia  has  been  entirely  removed.^  Collect  the  ammonia  in  a  100 
c.c.  volumetric  flask  containing  about  20  c.c.  of  ammonia-free  water  and 
2  c.c.  of  N/10  acid. 

'  With  any  given  filter  pump  a  "check"  test  should  be  made  with  urine  or  better  with  a 
solution  of  an  ammonium  salt  of  known  strength  to  determine  how  long  the  air  current  must 
be  maintained  to  remove  all  the  ammonia  from  25  c.c.  of  the  solution. 

2  Steele:  Jour.  Biol.  Chem.,  8,  365,  1910. 

*  Folin  and  Macallum:  Jour.  Biol.  Chem.  11,  523,  1912, 

*  The  volume  of  urine  taken  should  contain  0.75-1.5  mg.  of  ammonia  nitrogen.  With 
normal  urines  2  c.c.  will  generally  yield  the  desired  amount.  With  very  dilute  urines  5  c.c. 
may  be  required,  while  with  diabetic  urines  rich  in  ammonium  salts  i  c.c.  may  be  excessive, 
thus  requiring  dilution. 

*  Ordinarily  a  period  of  ten  minutes  is  sufficiently  long. 


urine:  quantitative  analysis.  401 

Nesslerize  as  described  in  the  method  for  total  nitrogen,  page  402,  and 
compare  with  i  mg.  of  nitrogen  obtained  from  a  standard  ammonium 
sulphate  solution  and  similarly  Nesslerized. 

It  has  been  noted  that  a  trace  of  something  capable  of  giving  a  color 
with  the  Nessler- Winkler  solution  continues  to  come  long  after  all  the 
ammonia  has  been  removed  from  the  urine.  The  nature  of  this  substance 
has  not  yet  bopn  determined.  In  actual  determinations,  by  this  method, 
the  influence  of  this  unknown  substance,  because  of  the  small  volume 
of  urine  used,  is  entirely  negligible. 

VI.  Total  Nitrogen. 

I.  Kjeldahl Method.^— The  principle  of  this  method  is  the  conversion 
of  the  various  nitrogenous  bodies  of  the  urine  into  ammonium  sulphate  by 
boiling  with  concentrated  sulphuric  acid,  the  subsequent  decomposition 
of  the  ammonium  sulphate  by  means  of  a  fixed  alkali  (NaOH)  and  the  col- 
lection of  the  liberated  ammonia  in  an  acid  of  known  strength.  Finally, 
this  partly  neutralized  acid  solution  is  titrated  with  an  alkali  of  known 
strength  and  the  nitrogen  content  of  the  urine  under  examination 
computed. 

The  procedure  is  as  follows:  Place  5  c.c.  of  urine  in  a  500  c.c.  long- 
necked  Jena  glass  Kjeldahl  flask,  add  20  c.c.  of  concentrated  sulphuric 
acid  and  about  0.2  gram  of  copper  sulphate  and  boil  the  mixture  for  some 
time  after  it  is  colorless  (about  one  hour).  Allow  the  flask  to  cool  and 
dilute  the  contents  with  about  200  c.c.  of  ammonia-free  water.  Add  a 
little  more  of  a  concentrated  solution  of  NaOH  than  is  necessary  to  neutral- 
ize the  sulphuric  acid"  and  introduce  into  the  flask  a  little  coarse  pumice 
stone  or  a  few  pieces  of  granulated  zinc,^  to  prevent  bumping,  and  a  small 
piece  of  paraffin  to  lessen  the  tendency  to  froth.  By  means  of  a  safety- 
tube  connect  the  flask  with  a  condenser  so  arranged  that  the  delivery-tube 
passes  into  a  vessel  containing  a  known  volume  (the  volume  used  depend- 
ing upon  the  nitrogen  content  of  the  urine)  of  N/io  sulphuric  acid,  using 
care  that  the  end  of  the  delivery-tube  reaches  beneath  the  surface  of  the 
fluid.*  Mix  the  contents  of  the  distillation  flask  very  thoroughly  by 
shaking  and  distil  the  mixture  until  its  volume  has  diminished  about  one- 
half.     Titrate  the  partly  neutralized  N/ 10  sulphuric  acid  solution  by 

'  There  are  numerous  modifications  of  the  original  Kjeldahl  method;  the  one  described 
here,  however,  has  given  excellent  satisfaction  and  is  recommended  for  the  determination  of 
the  nitrogen  content  of  urine. 

-  This  concentrated  sodium  hydroxide  solution  should  be  prepared  in  quantity  and  "check" 
tests  made  to  determine  the  voluine  of  the  solution  necessary  to  neutralize  the  volume  (20  c.c.) 
of  concentrated  sulphuric  acid  used. 

'  Powdered  zinc  may  be  substituted. 

*  This  deliver}-- tube  should  be  of  large  caliber  in  order  to  avoid  the   "sucking  back" 
of  the  lluid. 
26 


402  PHYSIOLOGICAL    CHEMISTRY. 

means  of  N/io  sodium  hydroxide,  using  congo  red  as  indicator,  and 
calculate  the  content  of  nitrogen  of  the  urine  examined. 

Calculation. — Subtract  the  number  of  cubic  centimeters  of  N/io 
sodium  hydroxide  used  in  the  titration  from  the  number  of  cubic  centi- 
meters of  N/io  sulphuric  acid  taken.  The  remainder  is  equivalent 
to  the  number  of  cubic  centimeters  of  N/io  sulphuric  acid,  neutralized 
by  the  ammonia  of  the  urine.  One  c.c.  of  N/io  sulphuric  acid  is  equivalent 
to  0.0014  gram  of  nitrogen.  Therefore,  if  }'  represents  the  volume  of 
urine  used  in  the  determination,  and  v'  the  number  of  cubic  centimeters 
of  N/io  sulphuric  acid  neutralized  by  the  ammonia  of  the  urine,  we  have 
the  following  proportion : 
Y  :  100  ::y'Xo. 0014  :  jc;  (percentage  of  nitrogen  in  the  urine  examined). 

Calculate  the  quantity  of  nitrogen  in  the  twenty-four-hour  urine 
specimen. 

Calculation  of  Percentage  Nitrogen  Distribution. — In  modern  metabol- 
ism studies  where  the  various  forms  of  nitrogen  are  determined,  in 
addition  to  the  total  nitrogen  as  yielded  by  the  Kjeldahl  method,  it  is 
customary  to  indicate  what  portion  of  the  total  nitrogen  was  present  in 
the  form  of  each  of  the  individual  nitrogenous  constituents.  These 
percentage  values  are  secured  by  dividing  the  weight  (grams)  of  nitrogen 
excreted  for  the  day  in  the  form  of  each  individual  nitrogenous  constituent 
by  the  weight  of  the  total  nitrogen  output  for  the  same  period.  For 
example,  if  the  total  nitrogen  excretion  is  9.814  grams  and  the  excretion 
of  urea-nitrogen  is  8.520  grams  and  the  excretions  of  nitrogen  in  the  forms 
of  ammonia  and  creatinine  are  0.271  gram  and  0.639  gram  respectively, 
the  percentage  distribution  for  these  forms  of  nitrogen  would  be  calculated 
as  follows: 

8 .  520  grams  urea-nitrogen  -^      9.814  grams  total  nitrogen     =   84.3   percent 

0.271  gram  ammonia-nitrogen     h-      g.  8 14  grams  total  nitrogen     =      2.7   percent 
0.639  gram  creatinine-nitrogen    ^     9,814  grams  total  nitrogen     =     6.5   percent 

2.  Kjeldahl -Folin-Farmer  Colorimetric  method.^ — This  method 
may  be  considered  as  a  microchemical  method  based  on  the  Kjeldahl- 
Gunning  process  for  decomposing  nitrogenous  materials  and  on  the 
methods  of  Nessler  and  of  Folin  for  the  determination  of  ammonia 
(see  page  399).  In  the  regular  Kjeldahl  procedure  30-100  mg.  of 
nitrogen  is  manipulated  whereas  in  this  modification  only  about  i  mg.  is 
utilized.     The  method  is  as  follows: 

Introduce  5  c.c.  of  urine  into  a  50  c.c.  volumetric  flask  if  the  specific 
gravity  of  the  urine  is  over  1018,  or  into  a  25  c.c.  flask  if  the  specific 
gravity  is  less  than  1018.'     Fill  the  flask  to  the  mark  with  distilled  water 

'  Folin  and  P'armer:    Jour.  Biol.  Chem.,  ir,  493,  1912. 

^  The  purpose  is  to  so  dilute  the  urine  that  i  c.c.  of  the  diluted  lluid  shall  contain  0.75-1.5 
mg.  of  nitrogen. 


urink:  quantitative  analysis. 


403 


and  invert  il  several  times  in  order  lo  guarantee  thorough  mixing. 
Transfer  one  cubic  centimeter*  of  the  diluted  urine  to  a  large  (20-25 
mm.  X  200  mm.)  Jena-glass  test-tube.  Add  to  this  i  c.c.  of  cencentrated 
sulphuric  acid,  i  gram  of  potassium  sulphate,  i  drop  of  5  per  cent  copper 
sulphate  solution  and  a  small,  clean,  c|uartz  pebble  or  glass  bead.  (The 
pebble  or  bead  is  added  to  prevent  bumping.)  Hoil  the  mixture  over  a 
micro-burner.'   for  about  six  minutes,   i.  e.,  about  two  minutes  after  the 


M_y 


UJ 


U 


Fig.  129.  Fig.  ijo. 

Figs.  129  and  130. — Forms  of  Apparatus  used  in  Methods  of  Folin  and  Associates  for 
Determinationof  Total  Nitrogen,  Urea  and  Ammonia.    {From  Jour.  Bio!.  Client. ,\o\.  ii,  iqi2.) 

mixture  has  become  colorless.  Allow  to  cool  until  the  digestion  mixture 
begins  to  become  viscous.  This  ordinarily  takes  about  three  minutes. 
but  in  any  event  the  mixture  must  not  be  permitted  to  solidify.  Add  about 
6  c.c.  of  water  (a  few  drops  at  a  time,  at  first,  then  more  rapidly)  to  pre- 
vent solidification.  To  this  acid  solution  add  an  excess  of  sodium 
hydroxide  (3  c.c.  of  a  saturated  solution  is  sufficient)  and  aspirate  the 
liberated  ammonia  by  means  of  a  rapid  air  current^  into  a  volumetric 

'  This  measurement  should  be  made  by  means  of  a  modified  Ostwald  pipette  (see  Ostwald- 
Luther:  Physiko-Lliemische  Messuiigen,  2d.  ed.,  p.  135).  Such  pipettes  may  be  obtained  from 
Eimer  and  Amend,  New  York. 

-  A  type  of  burner  which  has  proven  satisfactory  is  Eimer  and  Amend's  No.  2587. 

^  Either  a  vacuum  pump  or  compressed  air  or  a  force  pump  may  be  used.  The  com- 
pressed air  method  is  rather  the  more  convenient  inasmuch  as  the  ammonia  may  be  collected 
directly  in  a  volumetric  tiask.  Inasmuch  as  the  necks  of  such  tlasks  (100  c.c.)  are  not  large 
enough  to  permit  of  the  use  of  a  two-hole  rubber  stopper  when  suction  is  used,  the  ammonia 
should  be  collected  in  one  of  the  Jena  test-tubes  previously  described  which  contains  2  c.c.  of 
N/io  hydrochloric  acid  and  about  5  c.c.  of  ammonia-free  water.  The  ammonium  salt  is  then 
transferred  to  the  volumetric  flask  with  40-50  c.c.  of  water  and  Nesslerized  as  described. 


404  PHYSIOLOGICAL   CHEMISTRY, 

flask  (100  c.c.)  containing  about  20  c.c.  of  ammonia-free  water  and  2  c.c. 
of  N/io  hydrochloric  acid.  (See  Figs.  129  and  130,  p.  403.)  The  air 
current  should  be  only  moderately  rapid  for  the  first  two  minutes  but 
at  the  end  of  this  two-minute  period  the  current  should  be  run  at  its 
maximum  speed  for  an  interval  of  eight  minutes. 

Disconnect  the  flask,  dilute  the  contents  to  about  60  c.c.  with  am- 
monia-free water  and  dilute  similarly  i  mg.  of  nitrogen  in  the  form  of 
ammonium  sulphate^  in  a  second  volumetric  flask.  Nesslerize  both 
solutions  as  nearly  as  possible  at  the  same  time  with  5  c.c.  of  Nessler- 
Winkler  solution^  diluted,  immediately  before  using,  with  about  25  c.c. 
of  ammonia-free  water  to  avoid  turbidity.  Immediately  fill  the  two 
flasks  to  the  mark  with  ammonia-free  water,  mix  well  and  determine  the 
relative  intensity  of  the  two  colors  by  means  of  a  Duboscq  colorimeter.^ 

The  color  of  the  unknown  should  be  adjusted  to  that  of  the  standard 
both  from  above  and  below  the  level  of  the  latter.  The  matching  of  the 
colors  is  ordinarily  very  easy.  It  is  desirable  to  make  the  readings  by 
diffused  daylight  if  possible.  If  electric  light  must  be  used,  a  sheet  of 
smooth  white  paper  should  be  interposed  between  the  colorimeter  and  the 
source  of  light. 

Calculation. — The  reading  of  the  standard  divided  by  the  reading  of 
the  unknown  gives  the  nitrogen  in  milligrams  in  the  volume  oj  the  urine 
taken.     Calculate  the  total  nitrogen  output  for  the  twenty-four-period. 

VII.  Amino  Nitrogen. 

Van  Slyke's Method.^ — The  method  is  based  on  the  fact  that  nitrous 
acid  in  solution  spontaneously  decomposes  with  formation  of  nitric  oxide : 

2HNO,<riHN03  +  NO. 

'  Care  should  be  taken  to  secure  the  pure  salt.  All  ammonium  salts  contain  pyridine 
bases  which  titrate  like  ammonia  but  do  not  react  with  Nessler's  reagent.  Pure  ammonium 
sulphate  may  be  prepared  by  decomposing  a  high-grade  ammonium  salt  with  sodium  hydroxide 
and  passing  the  liberated  ammonia  into  pure  sulphuric  acid.  The  salt  is  then  pret  ipitated  by 
means  of  alcohol,  then  brought  into  solution  in  water  and  re-precipitated  by  alcohol.  The 
final  product  should  be  dried  in  a  desi?cator  over  sulphuric  acid.  Dr.  H.  L.  Emerson  of  Boston 
prepares  a  salt  which  is  ver)'  satisfactory  for  use  in  this  method. 

^Chem.  Zeit.,  1899,  p.  541.     The  Nessler- Winkler  solution  has  the  following  formula: 

Mercuric  iodide 10  grams. 

Potassium  iodide 5  grams. 

Sodium  hydro.xide 20  grams. 

Water 100  c.c. 

The  mercuric  iodide  is  rubbed  up  in  a  small  porcelain  mortar  with  water,  then  washed  into 
a  flask  and  the  potassium  iodide  added.  The  sodium  hydroxide  is  dissolved  in  the  remaining 
water  and  the  cooled  solution  added  to  the  above  mixture.  The  solution  cleared  by  standing 
is  preserved  in  a  dark  bottle. 

The  25  c.c.  portion  of  the  diluted  reagent  should  be  added  about  one-third  at  a  time  to  the 
contents  of  the  flask.  It  is  very  essential  that  the  dilution  of  the  reagent  takes  place  immediately 
preceding  its  use,  inasmuch  as  the  diluted  reagent  deteriorates  in  a  few  minutes  as  is  indicated 
by  the  formation  of  a  brick-red  precipitate.  Fortunately  the  reagent  does  not  decompose  in 
this  manner  in  the  presence  of  the  ammonium  salt. 

'  The  standard  may  be  set  at  any  desired  depth  but  a  very  satisfactory  depth  is  20  mm. 
The  depth  should  be  uniform  throughout  any  series  of  comparative  tests. 

*  Van  Slyke:  Jour.  Biol.  Chem.,  9,  185,  191 1. 


urine:  qu.\>s'titative  analysis. 


405 


This  reaction  is  utilized  in  displacing  all  the  air  of  the  apparatus  with 
nitric  oxide.  The  amino  solution  is  then  introduced,  evolution  of  nitrogen 
mixed  with  nitric  oxide  resulting.  The  oxide  is  absorbed  by  alkaline 
permanganate  solution,  and  the  pure  nitrogen  measured  in  a  special  gas 
burette  shown  in  the  figure. 

The  determination  of  amino  nitrogen  is  carried  out  as  follows: 
50  c.c.  of  the  total  day's  urine  are  measured  into  a  flask  and  2  c.c.  of  concen- 
trated acid  added.     The  acid  urine  is  then  placed  in  an  autoclave  and 


Fig.  131. — Van  Slyke's  .\iiiNO  Nitrogen  Appar.\tus. 


heated  to  175°  C.  under  pressure  for  an  hour  and  a  half.  After  hydrol- 
ysis is  complete,  a  few  drops  of  sodium  alizarin  sulphonate  are  added  as 
indicator  and  potassium  hydroxide  added  in  such  quantity  as  to  leave 
an  excess  of  the  reagent.  The  solution  is  then  boiled  20-30  minutes  in 
order  to  get  rid  of  all  the  ammonia.  After  boiling,  the  solution  is  approxi- 
mately neutralized  and  the  volume  made  up  exactly  to  50  c.c.  Ten  cubic 
centimeters  of  this  is  then  used  for  each  amino  acid  determination. 

The  10  c.c.  of  urine  treated  as  described  are  contained  in  the  burette 
(B)  of  the  van  Slyke  apparatus  (see  Fig.  131).  The  detailed  manipulation 
of  the  urine  in  order  to  determine  the  amino  acid  content  is  very  simple  to 
follow.     Into  the  reaction  chamber  (D)  one  pours  28  c.c.  of  sodium  ni- 


40(l  PHYSIOLOGICAL    CHEMISTRY. 

trite  solution  (30  grams  to  100  c.c.  of  water)  and  7  c.c.  of  glacial  acetic 
acid.  The  stopper  is  then  inserted  in  (D),  and  the  three-way  stop-cock  (c) 
opened  so  as  to  allow  the  gases  to  escape  into  the  air.  5  c.c.  of  water  are 
now  placed  in  vessel  (A)  and  allowed  to  run  into  (D)  so  as  to  expell  the  air 
remaining  in  the  apparatus.  The  cock  (c)  is  closed,  (a)  left  open  and  the 
solution  from  (D)  allowed  to  back  up  into  (A)  until  about  5  c.c.  are 
accumulated  there,  (D)  being  shaken  so  as  to  get  rid  of  any  air  dissolved 
in  the  interacting  solutions.  The  gases  are  again  expelled  as  described 
bv  opening  (c).  This  process  is  repeated  thus  washing  the  last  traces  of 
air  from  the  apparatus,  (a)  is  now  opened,  (c)  closed  and  about  25  c.c. 
of  the  solution  forced  into  (A)  by  the  pressure  of  the  nitric  oxide  formed  in 
(D).  The  cock  (c)  is  then  opened  so  that  the  gas  passes  into  the  burette 
(F),  (a)  closed,  and  the  10  c.c.  of  urine  run  into  (D).  After  the  reaction 
has  run  for  five  minutes,  (D)  is  shaken  and  the  remainder  of  the  gas 
forced  from  (D)  into  the  burette  by  allowing  the  liquid  in  (A)  to  run  into 
(D).  The  gases  are  then  run  from  the  burette  into  the  pipette  (H), 
the  latter  is  thoroughly  shaken,  till  no  more  gas  is  absorbed  by  the  alkaline 
permanganate  solution.^  The  pure  nitrogen  gas  is  run  back  into  the 
burette  and  measured.  The  temperature  of  the  gas  and  barometric 
pressure  are  recorded.  Blanks  should  be  run  and  the  slight  error  due  to 
the  formation  of  nitrogen  gas  and  oxygen  from  the  interaction  of  sodium 
nitrite  and  glacial  acetic  acid  accounted  for." 

Calculation. — As  the  reaction  doubles  the  amount  of  nitrogen  present 
as  amino  nitrogen,  the  volume  of  nitrogen  found  must  be  divided  by  2. 
The  results  are  expressed  in  milligrams  of  nitrogen. 

VIII.  Hippuric  Acid. 

Dakin's  Methods."^  Preliminary  Procedure. — ^Place  150  c.c.  (or 
more;  of  the  urine  under  examination  in  a  porcelain  evaporating  dish 
and  evaporate  almost  to  dryness  upon  a  water-bath.  Add  about  i  gram 
of  sodium  dihydrogen  phosphate,  about  25  grams  of  gypsum  (CaSO^, 
2H,Oj  and  rub  up  with  a  pestle  and  stir  with  a  spatula  until  a  uniform 
mixture  results.  Dry  the  powder  thus  produced  in  a  water-oven  for 
about  two  hours,  at  the  end  of  which  period  it  should  be  rubbed  up  a 
second  time,  to  remove  lumps,  and  transferred  to  a  Schleicher  and  Schiill 
"extraction  shell"  and  extracted  in  a  Soxhlet  apparatus  in  the  usual  way 

'  The  alkaline  permanKanale  solution  contains  50  grams  of  potassium  permanganate  and 
25  grams  of  jxjtassium  hyflroxide  per  liter. 

^According  to  Robinson  (Mich.  Ag.  Exp.  Station  Tech.  Bull.,  7,  p.  11,  1911),  analysis 
of  the  gases  formed  by  the  decomposition  of  sodium  nitrite  with  glacial  acetic  acid  indicates 
the  presence  of  small  amounts  of  free  oxygen  and  nitrogen. 

'Private  communifation  to  the  author  from  Dr.  H.  D.  Dakin. 


urine:  quantitative  analysis.  407 

(see  p.  437).  The  extraction  medium  is  ethyl  acetate  and  the  flask  con- 
taining the  acetate  should  be  strongly  heated  over  a  sand-bath^  for  about 
two  hours.  The  ethyl  acetate  extract  is  now  transferred  to  a  separatory 
funnel,  and  the  original  flask  rinsed  with  sufficient  fresh  ethyl  acetate 
to  make  the  total  volume  in  the  separatory  funnel'-'  about  100  c.c.  Wash 
the  ethyl  acetate  solution  //ir  times  with  a  saturated  solution  of  sodium 
chloride,  usijig  8  c.c.  of  the  sodium  chloride  solution  at  each  extraction, 
shaking  vigorously  and  removing  the  sodium  chloride  extract  in  each  case 
before  adding  fresh  sodium  chloride  solution.  The  sodium  chloride 
removes  the  urea  completely  and  the  hippuric  acid  is  then  determined  in  the 
urea-free  solution  by  the  following  volumetric  or  gravimetric  procedure: 

1.  Volumetric  Determination. — Transfer  the  urea-free  ethyl  acetate 
solution,  prepared  as  described  above,  to  a  Kjeldahl  flask,  add  about 
25  c.c.  of  water,  a  small  piece  of  pumice  stone  to  prevent  bumping, 
attach  a  condenser  and  distil  off  the  acetate''  over  a  free  flame.  After 
practically  all  of  the  ethyl  acetate  has  been  distilled  off,  the  nitrogen  in  the 
remaining  solution  should  be  determined  by  means  of  the  Kjeldahl 
method  (see  p.  402). 

The  main  source  of  error  in  this  method  is  the  fact  that  any  nitrogen 
present  in  the  form  of  phenaceturic  acid  or  indole  acetic  acid  is  determined 
as  hippuric  acid  nitrogen.  The  error  from  this  source  is,  however,  usually 
trifling. 

Calculatim. — Calculate  as  usual  for  nitrogen  determinations,  re- 
membering that  I  c.c.  oj  N/io  sulphuric  acid  is  equivalent  to  0.0 17Q  gram 
hippuric  acid. 

2.  Gravimetric  Determination. — The  urea-free  ethyl  acetate  solu- 
tion, contained  in  the  separatory  funnel,  after  washing  with  sodium 
chloride  solution,  as  described  under  Preliminary  Procedure,  p.  406, 
is  washed  with  5  c.c.  of  distilled  water  to  remove  the  major  portion  of 
the  sodium  chloride.  Transfer  the  solution  from  the  separatory  funnel 
to  a  round-bottomed  flask  and  subject  it  to  a  steam  distillation  in  the 
usual  way.  A  slow  current  of  steam  should  be  used  while  the  ethyl 
acetate  is  being  distilled  off  and  later  a  more  rapid  current  may  be  em- 
ployed. The  distillation  should  be  continued  for  twenty  minutes.  Now 
add  about  o.i  gram  of  charcoal  to  the  aqueous  solution  which  is  heated 
to  boiling  and  filtered  hot.  Evaporate  the  solution  in  a  weighed  Jena 
glass  dish  on  a  water-bath  until  the  volume  of  the  solution  is  reduced  to 
about  3  c.c.     Stand  the  dish  in  a  warm  place  until  evaporation  is  complete 

'  A  water-bath  cannot  be  substituted  inasmuch  as  the  resuUant  extraction  would  be  too 
slow. 

-  This  ethyl  acetate  solution  contains  hippuric  acid,  urea,  and  other  substances. 

^  The  ethyl  acetate  after  separation  from  the  watef}'  layer  of  the  distillate  may  be  dried 
over  calcium  chloride  and  used  agian. 


4o8  PHYSIOLOGICAL    CHEMISTRY. 

and  a  crystalline  residue  remains.  Wash  the  residue,  in  turn,  with  2 
c.c.  of  dry  ether,  and  i  c.c.  of  water,  dry  it  in  an  air-bath  at  100°  C.  and 
weigh.  If  it  is  so  desired  the  residue  may  be  recrystallized  from  a  little 
hot  water  and  the  melting-point  determined.  Pure  hippuric  acid  melts  at 
187°  C.  Contamination  with  phenaceturic  acid  may  be  detected  both  by 
the  melting-point  and  the  microscopical  characteristics. 

IX.  Sulphur. 

I.  Total  Sulphates.  Faun's  Method. — ^Place  25  c.c.  of  urine  in  a 
200-250  c.c.  Erlenmeyer  flask,  add  20  c.c.  of  dilute  hydrochloric  acid^ 
(i  volume  of  concentrated  HCl  to  4  volumes  of  water)  and  gently  boil  the 
mixture  for  20-30  minutes.  To  niinimize  the  loss  of  water  by  evaporation 
the  mouth  of  the  flask  should  be  covered  with  a  small  watch  glass  during 
the  boiling  process.  Cool  the  flask  for  2-3  minutes  in  running  water, 
and  dilute  the  contents  to  about  150  c.c.  by  means  of  cold  water.  Add 
10  c.c.  of  a  5  per  cent  solution  of  bariuni  chloride  slowly,  drop  by  drop,  to 
the  cold  solution.^  The  contents  of  the  flask  should  not  he  stirred  or 
shaken  during  the  addition  of  the  barium  chloride.  Allow  the  mixture  to 
stand  at  least  one  hour,  then  shake  up  the  solution  and  filter  it  through 
a  weighed  Gooch  crucible.^ 

Wash  the  precipitate  of  BaSO^  with  about  250  c.c.  of  cold  water, 
dry  it  in  an  air-bath  or  over  a  very  low  flame,  then  ignite,*  cool  and  weigh. 

Calculation. — Subtract  the  weight  of  the  Gooch  crucible  from  the 
weight  of  the  crucible  and  the  BaSO^  precipitate  to  obtain  the  weight  of 
the  precipitate.  The  weight  of  Sog^  in  the  volume  of  urine  taken  may 
be  determined  by  means  of  the  following  proportion. 

Mol.  wt.       Wt.  of  Mol.  wt. 

BaSO^ :  BaSO^ : :  SO3  -.x  (wt.  of  SO3  in  grams). 

*  If  it  is  desired,  50  c.c.  of  urine  and  4  c.c.  of  concentrated  acid  may  be  used  instead. 

^  A  dropper  or  capillary  funnel  made  from  an  ordinar)-  calcium  chloride  tube  and  so 
constructed  as  to  deliver  10  c.c.  in  2-3  minutes  is  recommended  for  use  in  adding  the  barium 
chloride. 

^  If  a  Gooch  crucible  is  not  available,  the  precipitate  of  BaS04  may  be  filtered  off  upon 
a  washed  filter  paper  (Schleicher  &  Schiill's,  No.  589,  blue  ribbon),  and  after  washing  the 
precipitate  with  about  250  c.c.  of  cold  water  the  paper  and  precipitate  may  be  dried  in  an 
air-bath  or  over  a  low  (lame.  The  ignition  may  then  be  carried  out  in  the  usual  way  in  the 
ordinar}'  platinum  or  porcelain  crucible.  In  this  case  correction  must  be  made  for  the  weight 
of  the  ash  of  the  filter  paper  used. 

''  Care  must  be  taken  in  the  ignition  of  precipitates  in  Gooch  crucibles.  The  flame 
should  never  be  applied  directly  to  the  perforated  bottom  or  to  the  sides  of  the  crucible,  since 
such  manipulation  is  invariably  attended  by  mechanical  losses.  The  crucibles  should  always 
be  provided  with  lids  and  tight  bottoms  during  the  ignition.  In  case  porcelain  Gooch  crucibles, 
whose  bottoms  are  not  provided  with  a  non-perforated  cap,  are  used,  the  crucible  may  be 
placed  upon  the  lid  of  an  ordinary  platinum  crucible  during  ignition.  The  lid  should  be 
supported  on  a  triangle,  the  crucible  placed  upon  the  lid  and  the  flame  applied  to  the  im- 
provised bottom.     Ignition  should  be  complete  in  10  minutes  if  no  organic  matter  is  present. 

*  It  is  considered  preferable  by  many  investigators  to  express  all  sulphur  values  in  terms 
of  S  rather  than  SO,. 


urine:  quantitative  analysis.  409 

Representing  the  weight  of  the  BaSO^  precipitate  by  y  and  substituting 
the  proper  molecular  weights,  we  have  the  following  proportion : 
231.7:^:  :79.5  r.Y   (wt.  of  SO3  in  grams  in  the  quantity  of  urine  used). 

Calculate  the  quantity  of  SO3  in  the  twenty-four-hour  specimen  of 
urine. 

To  express  the  result  in  percentage  of  SO3  simply  divide  the  value  of  x, 
as  just  determined,  by  the  quantity  of  urine  used. 

2.  Inorganic  Sulphates.  Folin's  Method. — Place  25  c.c.  of  urine 
and  100  c.c.  of  water  in  a  200-250  c.c.  Erlenmeyer  flask  and  acidify  the 
diluted  urine  with  10  c.c.  of  dilute  hydrochloric  acid  (i  volume  of  con- 
centrated HCl  to  4  volumes  of  water).  In  case  the  urine  is  dilute  50  c.c. 
may  be  used  instead  of  25  c.c.  and  the  volume  of  water  reduced  pro- 
portionately. Add  10  c.c.  of  5  per  cent  barium  chloride  slowly,  drop  by 
drop,  to  the  cold  solution  and  from  this  point  proceed  as  indicated  in  the 
method  for  the  determination  of  Total  Sulphates,  page  408. 

Calculate  the  quantity  of  inorganic  sulphates,  expressed  as  SO3,  in 
the  twenty-four-hour  urine  specimen. 

Calculation. — Calculate  according  to  the  directions  given  under  Total 
Sulphates,  above. 

3.  Ethereal  Sulphates.  Folin's  Method. — Place  125  c.c.  of  urine  in 
an  Erlenmeyer  flask  of  suitable  size,  dilute  it  with  75  c.c.  of  water  and 
acidify  the  mixture  with  30  c.c.  of  dilute  hydrochloric  acid  (i  volume  of 
concentrated  HCl  to  4  volumes  of  water).  To  the  coW  solution  add  20  c.c. 
of  a  5  per  cent  solution  of  barium  chloride,  drop  by  drop.^  Allow  the 
mixture  to  stand  about  one  hour,  then  filter  it  through  a  dry  filter  paper.^ 
Collect  125  c.c.  of  the  filtrate  and  boil  it  gently  for  at  least  one-half  hour. 
Cool  the  solution,  filter  off  the  precipitate  of  BaSO^,  wash,  dry  and  ignite 
it  according  to  the  directions  given  on  page  408. 

Calcidation. — The  weight  of  the  BaSO^  precipitate  should  be  multi- 
plied by  2  since  only  one-half  (125  c.c.)  of  the  total  volume  (250  c.c.)  of 
fluid  was  precipitated  by  the  barium  chloride.  The  remaining  calculation 
should  be  made  according  to  directions  given  under  Total  Sulphates,  page 
408. 

Calculate  the  quantity  of  ethereal  sulphates,  expressed  as  SO 3,  in  the 
twenty-four-hour  urine  specimen. 

4.  Total  Sulphur.  Benedict's  Method.^— Ten  cubic  centimeters  of 
urine  are  measured  into  a  small  (7-8  cm.)  porcelain  evaporating  dish  and 

'  See  note  (2)  at  the  bottom  of  page  408. 

*  This  precipitate  consists  of  the  inorganic  sulphates.  If  it  is  desired,  this  BaSO^  pre- 
cipitate may  be  collected  in  a  Gooch  crucible  or  on  an  ordinary  quantitative  filter  paper  and  a 
determination  of  inorganic  sulphates  made,  using  the  same  technic  as  that  suggested 
above.  In  this  way  we  are  enabled  to  determine  the  inorganic  and  ethereal  sulphates 
in  the  same  sample  of  urine. 

'  Benedict:   Journal  of  Biological  Chemistry,  6,  363,  1909. 


4IO  PHYSIOLOGICAL    CHEMISTRY. 

5  c.c.^  of  Benedict's  sulphur  reagent"  added.  The  contents  of  the  dish 
are  evaporated  over  a  free  flame  which  is  regulated  to  keep  the  solution 
just  below  the  boiling-point,  so  that  there  can  be  no  loss  through  spattering. 
When  dryness  is  reached  the  flame  is  raised  slightly  until  the  entire 
residue  has  blackened.  The  flame  is  then  turned  up  in  two  stages  to  the 
full  heat  of  the  bunsen  burner  and  the  contents  of  the  dish  thus  heated  to 
redness  for  ten  minutes  after  the  black  residue  {which  first  fuses)  has- become 
dry.  This  heating  is  to  decompose  the  last  traces  of  nitrate  (and  chlorate) . 
The  flame  is  then  removed  and  the  dish  allowed  to  cool  more  or  less 
completely.  Ten  to  twenty  cubic  centimeters  of  dilute  (1:4)  hydrochloric 
acid  is  then  added  to  the  residue  in  the  dish,  which  is  then  warmed  gently 
until  the  contents  have  completely  dissolved  and  a  perfectly  clear,  spark- 
ling solution  is  obtained.  This  dissolving  of  the  residue  requires  scarcely 
two  minutes.  With  the  aid  of  a  stirring  rod  the  solution  is  washed  into^ 
a  small  Erlenmeyer  flask,  diluted  with  cold,  distilled  water  to  100-150  c.c, 
10  c.c.  of  10  per  cent  barium  chloride  solution  added  drop  by  drop,  and 
the  solution  allowed  to  stand  for  about  an  hour.  It  is  then  shaken  up 
and  filtered  as  usual  through  a  weighed  Gooch  crucible. 

Calculation. — Make  the  calculation  according  to  directions  given  under 
Total  Sulphates,  p.  408.  Calculate  the  quantity  of  sulphur,  expressed 
as  SO3  or  S,  present  in  the  twenty-four-hour  urine  specimen. 

5.  Total  Sulphur.  Osborne-Folin  Method. — ^Place  25  c.c.  of  urine* 
in  a  200-250  c.c.  nickel  crucible  and  add  about  3  grams  of  sodium  perox- 
ide. Evaporate  the  mixture  to  a  syrup  upon  a  steam  water-bath  and 
heat  it  carefully  over  an  alcohol  flame  untfl  it  solidifies  (15  minutes). 
Now  remove  the  crucible  from  the  flame  and  allow  it  to  cool.  Moisten 
the  residue  with  1-2  c.c.  of  water,^  sprinkle  about  7-8  grams  of  sodium 
peroxide  over  the  contents  of  the  crucible  and  fuse  the  mass  over  an 
alcohol  flame  for  about  10  minutes.  Allow  the  crucible  to  cool  for  a  few 
minutes,  add  about  100  c.c.  of  water  to  the  contents  and  heat  at  least 
one-half  hour  over  an  alcohol  flame  to  dissolve  the  alkali  and  decompose 
the  sodium  peroxide.  Next  rinse  the  mixture  into  a  400-450  c.c.  Erlen- 
meyer flask,  by  means  of  hot  water,  and  dilute  it  to  about  250  c.c.  Heat 
the  solution  nearly  to  the  boiling-point  and  add  concentrated  hydrochloric 
acid  slowly  until  the  nickelic  oxide,  derived  from  the  crucible,  is  just 

'  If  llif  urine  is  concentratefi  the  ([uanlity  shouM  be  slightly  increased. 

-  Crystallizetl  copper  nitrate,  sulphur-free  or  of  known  sul])hur  content...  .     200  grams. 

Soflium  or  potassium  chlorate 50  grams. 

Distilled  water  to 1000  C.C. 

■'*  .Sometimes  the  ]>orcelain  glaze  cracks  during  heating,  in  which  case  the  solution  should 
be  filtered  into  the  flask. 

■*  If  the  urine  is  very  dilute  50  c.c.  may  be  used. 

^  This  moistening  of  the  residue  with  a  small  amount  of  water  is  very  essential  and  should 
not  be  neglected. 


URINK:    (PUANTITATIVE   ANALYSIS. 


411 


brcnight  into  solution.'  A  few  minutes  boiling  should  now  yield  a  clear 
solution.  In  case  too  little  peroxide  or  too  much  water  was  added  for  the 
final  fusion  a  clear  solution  will  not  be  obtained.  In  this  event  cool  the 
solution  and  remove  the  insoluble  matter  bv  filtration. 


Fig.  132. — Bertheldt-Atwatek  Ishmb  c  aiurimeter.     K  r<)ss-secti(in  ok  Appar-atcs  as 

Ready  for  Use.) 
.\,  Steel  cup  or  bomb  proper;  C,  collar  of  steel;  G,  opening  through  which  o.wgen  is  forcefi 
into  the  bomb;  H  and  I',  insulated  wires  which  serve  to  conduct  an  electric  current  for  igniting 
the  substance  which  is  held  in  the  small  capsule;  L,  a  stirrer  which  serves  to  keep  the  water 
surrounding  the  bomb  in  motion  and  insures  the  equalization  of  temperature;  P,  a  delicate 
thermometer  which  shows  the  rise  in  temperature  of  the  water  surrounding  the  bomb. 

To  the  clear  solution  add  5  c.c.  of  very  dilute  alcohol  (about  18-20 
per  cent)  and  continue  the  boiling  for  a  few  minutes.  The  alcohol  is 
added  to  remove  the  chlorine  which  was  formed  when  the  solution  was 

•  .\bout  18  c.c.  of  acid  is  rec|uired  for  8  grams  of  sodium  {)eroxide. 


412  PHYSIOLOGICAL   CHEMISTRY. 

acidified.  Add  lo  c.c.  of  a  lo  per  cent  solution  of  barium  chloride,  slowly, 
drop  by  drop,^  to  the  liquid.  Allow  the  precipitated  solution  to  stand  in 
the  cold  two  days  and  then  filter  and  continue  the  manipulation  according 
to  the  directions  given  under  Total  Sulphates,  page  408. 

Calculation. — Make  the  calculation  according  to  directions  given 
under  Total  Sulphates,  p.  408.  Calculate  the  quantity  of  sulphur, 
expressed  as  SO3  or  S,  present  in  the  twenty-four-hour  urine  specimen. 

6.  Total  Sulphur. — Sodium  Hydroxide  and  Potassium  Nitrate  Fusion 
Method. — ^Place  25  c.c.  of  urine  in  a  silver  crucible  and  evaporate  to  a 
thick  syrup  on  a  water-bath.  Add  10  grams  of  sodium  hydroxide  and  2 
grams  of  potassium  nitrate  to  the  residue  and  fuse  the  mass,  over  an 
alcohol  flame,  until  all  organic  matter  has  disappeared  and  the  fused 
mixture  is  clear.  Cool  the  mixture,  transfer  it  to  a  casserole  by  means  of 
hot  water,  acidify  slightly  with  hydrochloric  acid  and  evaporate  it  to  dry- 
ness on  a  water-bath.  Moisten  the  residue  with  a  few  drops  of  dilute 
hydrochloric  acid  and  bring  it  into  solution  with  hot  water.  Filter,  heat 
the  filtrate  to  boiling,  and  immediately  precipitate  it  by  the  addition  of  10 
c.c.  of  a  10  per  cent  solution  of  barium  chloride,  adding  the  solution 
slowly,  drop  by  drop.  Allow  the  precipitated  solution  to  stand  2  hours 
and  filter  while  cold.  Ignite,  weigh,  and  calculate  according  to  directions 
given  under  Total  Sulphates,  p.  408. 

Compute  the  quantity  of  sulphur,  expressed  as  SO3  or  S,  present  in 
the  twenty-four-hour  urine  specimen. 

7.  Total  Sulphur.  Sherman's  Compressed  Oxygen  Method."^ — 
Evaporate  as  much  urine  on  an  absorbent  filter  block^  at  55°  C.  as  the 
block  will  conveniently  absorb  and  burn  the  block  so  prepared  in  a  bomb- 
calorimeter^  using  25-30  atmospheres  of  oxygen.  Connect  the  bomb 
with  a  wash-bottle  containing  water,  and  allow  the  gas  to  bubble  through 
the  liquid  until  the  high  pressure  within  the  apparatus  has  been  reduced 
to  atmospheric  pressure.  Now  open  the  bomb  and  thoroughly  rinse  the 
interior,  using  water  from  the  wash-bottle  for  the  first  rinsing.  Dissolve 
any  ash  found  in  the  combustion  capsule  in  hydrochloric  acid  and  add 
this  solution  to  the  main  solution.  Evaporate  to  150  c.c,  filter,  and  cool 
the  filtrate.  Add  10  c.c.  of  a  5  per  cent  solution  of  barium  chloride  to  the 
cold  filtrate,  slowly,  drop  by  drop.^  The  contents  of  the  flask  should  not 
be  stirred  or  shaken  during  the  addition  of  the  barium  chloride.  Allow 
the  mixture  to  stand  at  least  one  hour,  then  shake  up  the  solution  and 

'  See  note  (2)  at  the  bottom  of  page  408. 

^  See  Sherman's  Organic  Analysis,  First  edition,  p.  19. 

^  Only  a  small  amount  of  urine  should  be  added  at  one  time,  it  being  necessary  to  make 
several  evaporations  before  the  block  contains  sufficient  urinary  residue  to  proceed  with  the 
combustion. 

*  The  Berthelot-Atwater  apparatus  (Fig.  132.  page  411)  is  well  adapted  to  this  purpose. 

*  See  note  (2)  at  the  bottom  of  page  408. 


urine:  quantitative  analysis.  413 

filter  il  throuj^h  a  weighed  Gooch  crucible.     Manipulate  the  precipitate 
of  BaSO^  according  to  directions  given  under  Total  Sulphates,  page  408. 

Calculate  the  quantity  of  sulphur,  expressed  as  SO3  or  S,  present  in  the 
twenty-four-hour  urine  specimen. 

X.  Phosphorus. 

1.  Total  Phosphates.  Uranium  Acetate  Method. — To  50  c.c.  of 
urine  in  a  small  beaker  or  Erlenmeyer  flask  add  5  c.c.  of  a  special  sodium 
acetate  solution'  and  heat  the  mixture  to  the  boiling-point.  From  a 
burette,  run  into  the  hot  mixture,  drop  by  drop,  a  standard  solution  of 
uranium  acetate"  until  a  precipitate  ceases  to  form  and  a  drop  of  the  mix- 
ture when  removed  by  means  of  a  glass  rod  and  brought  in  contact  with  a 
drop  of  a  solution  of  potassium  ferrocyanide  on  a  porcelain  test-tablet  pro- 
duces instantaneously  a  brownish-red  coloration.^  Take  the  burette 
reading  and  calculate  the  PjOg  content  of  the  urine  under  examination. 

Cakulation. — Multiply  the  number  of  cubic  centimeters  of  uranium 
acetate  solution  used  by  0.005  to  determine  the  number  of  grams  of  PjOg 
in  the  50  c.c.  of  urine  used.  To  express  the  result  in  percentage  of  P2O5 
multiply  the  value  just  obtained  by  2,  e.  g.,  if  50  c.c.  of  urine  contained 
0.074  gram  of  PoOj  it  would  be  equivalent  to  0.148  per  cent. 

Calculate,  in  terms  of  PjOj,  the  total  phosphate  content  of  the  twenty- 
four-hour  urine  specimen. 

2.  Earthy  Phosphates. — To  100  c.c.  of  urine  in  a  beaker  add  an 
excess  of  ammonium  hydroxide  and  allow  the  mixture  to  stand  12-24 
hours.  Under  these  conditions  the  phosphoric  acid  in  combination  with 
the  alkaline  earths,  calcium  and  magnesium,  is  precipitated  as  phosphates 
of  these  metals.  Collect  the  precipitate  on  a  filter  paper  and  wash  it 
with  very  dilute  ammonium  hydroxide.  Pierce  the  paper,  and  remove  the 
precipitate  by  means  of  hot  water.  Bring  the  phosphates  into  solution  by 
adding  a  small  amount  of  dilute  acetic  acid  to  the  warm  solution.  Make 
the    volume    up   to  50  c.c.  with  water,  add  5  c.c.  of  sodium  acetate 

'  The  sodium  acetate  solution  is  prepared  bv  dissolving  loo  grams  of  sodium  acetate 
in  Soo  c.c.  of  distilled  water,  adding  loo  c.c.  of  30  per  cent  acetic  acid  to  the  solution,  and 
making  the  volume  of  the  mixture  up  to  r  liter  with  water. 

-  This  uranium  acetate  solution  may  be  prepared  by  dissolving  about  34.  grams  of  uranium 
acetate  in  one  liter  of  water.  One  c.c.  of  this  solution  should  now  be  made  equivalent  to 
0.005  gram  of  PjO,,  phosphoric  anhydride.  It  may  be  standardized  as  follows:  To  50  c.c. 
of  a  standard  solution  of  disodium  hydrogen  phosphate,  of  such  a  strength  that  the  50  c.c. 
contains  o.i  gram  of  PoO^,  add  5  c.c.  of  the  sodium  acetate  solution  mentioned  above,  and 
titrate  with  the  uranium  solution  to  the  correct  end-reaction  as  indicated  in  the  method  proper. 
Inasmuch  as  i  c.c.  of  the  uranium  solution  should  precipitate  0.005  gr^i^"'  '^^  P20,-„  exactly 
20  c.c.  of  the  uranium  solution  should  be  required  to  precipitate  50  c.c.  of  the  standard  phos- 
phate solution.  If  the  two  solutions  do  not  bear  this  raeltion  to  each  other  they  may  be 
brought  into  pro[)cr  relation  by  diluting  the  uranium  solution  with  distilled  water  or  by  in- 
creasing its  strength. 

'  .\  lo  per  cent  solution  of  potassium  ferrocyanide  is  satisfactory. 


414  PHYSIOLOGICAL    CHEMISTRY. 

solution,  and  determine  the  P20g  content  of  the  mixture  according  to  the 
directions  given  under  the  previous  method. 

Calculation. — Multiply  the  number  of  cubic  centimeters  of  uranium 
acetate  solution  used  by  0.005  to  determine  the  number  of  grams  of  P2O5 
in  the  100  c.c.  of  urine  used.  Since  100  c.c.  of  urine  was  taken  this  value 
also  expresses  the  percentage  of  P2O5  present. 

Calculate  the  quantity  of  earthy  phosphates,  in  terms  of  P2O5,  present 
in  the  twenty-four-hour  urine  specimen. 

The  quantity  of  phosphoric  acid  present  in  combination  with  the 
alkali  metals  may  be  determined  by  subtracting  the  content  of  earthy 
phosphates  from  the  total  phosphates. 

Total  Phosphorus. — Sodium  Hydroxide  and  Potassium  Nitrate 
Fusion  Method.— Vlsice  25  c.c.  of  urine  in  a  large  silver  crucible  and  evapo- 
rate to  a  syrup  on  a  water-bath.  Add  10  grams  of  NaOH  and  2  grams  of 
KNO3  to  the  residue  and  fuse  the  mass  until  all  organic  matter  has  dis- 
appeared and  the  fused  mixture  is  clear.  Cool  the  mixture,  transfer  it  to 
a  casserole  by  means  of  hot  water,  acidify  the  solution  slightly  with  pure 
nitric  acid,  and  evaporate  to  dryness  on  a  water-bath.  Moisten  the 
residue  with  a  few  drops  of  dilute  nitric  acid,  dissolve  it  in  hot  water,  and 
transfer  to  a  beaker.  Now  add  an  equal  volume  of  molybdic  solution^  and 
keep  the  mixture  at  40°  C.  for  twenty-four  hours.  Filter  off  the  precipi- 
tate, wash  it  with  dilute  molybdic  solution,  and  dissolve  it  in  dilute 
ammonia.  Add  dilute  hydrochloric  acid  to  the  solution,  being  careful  to 
leave  the  solution  distinctly  ammoniacal.  Magnesia  mixture^  (lo-i  5  c.c.) 
should  now  be  added  and  after  stirring  thoroughly  and  making  strongly 
ammoniacal  with  concentrated  ammonia  the  solution  should  be  allowed 
to  stand  in  a  cool  place  for  twenty-four  hours.  Filter  off  the  precipitate, 
wash  it  free  from  chlorine  by  means  of  dilute  ammonia  (1:5),  dry,  inciner- 
ate, and  weigh,  as  magnesium  pyrophosphate,  Mg2P207,  in  the  usual 
manner. 

In  this  method  the  phosphoric  acid  of  the  urine  is  precipitated  as 
ammonium  magnesium  phosphate  and  in  the  process  of  incineration  this 
body  is  transformed  into  magnesium  pyrophosphate. 

Calculation. — The  quantity  of  phosphorus,  expressed  in  terms  of 
PjOj,  in  the  volume  of  urine  taken  may  be  determined  by  means  of  the 
following  proportion : 

.Mol.  wt.  Wt.  of  Mol.    wt. 

Mg,P,0,:Mg,P,0,::PA:^  (wt.  of  PA  in  grams). 
ppt. 


'  Directions  for  the  preparation  of  tlie  solution  are  given  on  p.  64. 

^  Directions  for  the  preparation  of  magnesia  mixture  may  be  found  on  p.  313. 


urine:  quantitative  analysis.  415 

If  V  represents  the  weight  of  the  Mg^PjO^  precipitate  and  we  make 
the  proper  substitution  we  have  the  following  proportion : 
221.1 :  V  :  :i40.9  :.v  (wt.  of  P^Oj.  in  grams,  in  the  ciuantity  of  urine  used.) 

To  express  the  result  in  percentage  of  FjOj  simply  divide  the  value  of 
-v.  as  just  determined,  by  the  quantity  of  urine  used. 

XI.    Creatinine. 

Folin's  Colorimetric  Method.— This  method  is  based  upon  the 
characteristic  property  possessed  alone  by  creatinine,  of  yielding  a 
certain  definite  color-reaction  in  the  presence  of  picric  acid  in  alkaline 
solution.  The  procedure  is  as  follows:  Place  10  c.c.  of  urine  in  a  500 
c.c.  volumetric  flask,  add  15  c.c.  of  a  saturated  solution  of  picric  acid 
and  5  c.c.  of  a  10  per  cent  solution  of  sodium  hydro.xidc.  shake  thoroughly 
and  allow  the  mixture  to  stand  for  5  minutes.  During  this  interval 
pour  a  little  N/2  potassium  bichromate  solution^  into  each  of  the 
two  cylinders  of  the  colorimeter  (Duboscq's)  and  carefully  adjust  the 
depth  of  the  solution  in  one  of  the  cylinders  to  the  8  mm.  mark.  A 
few  preliminary  colorimetric  readings  may  now  be  made  with  the 
solution  in  the  other  cylinder,  in  order  to  insure  greater  accuracy  in 
the  subsequent  examination  of  the  solution  of  unknown  strength. 
Obviously  the  two  solutions  of  potassium  bichromate  are  identical  in 
color  and  in  their  examination  no  two  readings  should  differ  more 
than  0.1-0.2  mm.  from  the  true  value  (8  mm.).  Four  or  more  readings 
should  be  made  in  each  case  and  an  average  taken  of  all  of  them  exclusive 
of  the  first  reading,  which  is  apt  to  be  less  accurate  than  the  succeeding 
readings.  In  time  as  one  becomes  proficient  in  the  technic  it  is 
perfectly  safe  to  take  the  average  of  \.\v€:  first  two  readings. 

At  the  end  of  the  5-minute  interval  already  mentioned,  the  contents 
of  the  500  c.c.  flask  are  diluted  to  the  500  c.c.  mark,  the  bichromate 
solution  is  thoroughly  rinsed  out  of  one  of  the  cylinders,  and  replaced 
with  the  solution  thus  prepared  and  a  number  of  colorimetric  readings 
are  immediately  made. 

Ordinarily  10  c.c.  of  urine  is  used  in  the  determination  by  this  method, 
but  if  the  content  of  creatinine  is  above  15  mg.  or  below  5  mg.  the  determi- 
nation should  be  repeated  with  a  volume  of  urine  selected  according  to 
the  content  or  creatinine.  This  vaiiation  in  the  volume  of  urine  according 
to  the  content  of  creatinine  is  quite  essential,  since  the  method  loses  in 
accuracy  when  more  than  15  mg.  or  less  than  5  mg.  of  creatinine  is 
present  in  the  solution  of  unknown  strength. 

Calculation. — By  experiment  it  has  been  determined  that    10  mg. 

'  This  solution  contains  24.55  grams  of  potassium  bichromate  to  the  liter. 


4l6  PHYSIOLOGICAL   CHEMISTRY. 

of  pure  creatinine,  when  brought  into  solution  and  diluted  to  500  c.c. 
as  explained  in  the  above  method,  yields  a  mixture  8.1  mm,  of  which 
possesses  the  same  colorimetric  value  as  8  mm.  of  a  N/2  solution  of 
potassium  bichromate.  Bearing  this  in  mind  the  computation  is  readily 
made  by  means  of  the  following  proportion  in  which  y  represents  the 
number  of  millimeters  of  the  solution  of  unknown  strength  equivalent  to 
the  8  mm.  of  the  potassium  bichromate  solution: 

}'  :  8.1  :  :  10  :  x  (mgs.  of  creatinine  in  the  quantity  of  urine  used). 

This  proportion  may  be  used  for  the  calculation  no  matter  what 
volume  of  urine  (5,  10,  or  15  c.c.)  is  used  in  the  determination.  The 
10  represents  10  mg.  of  creatinine  which  gives  a  color  equal  to  8.1  mm., 
whether  dissolved  in  5,  10,  or  15  c.c.  of  fluid. 

Calculate  the  quantity  of  creatinine  in  the  twenty-four-hour  urine 
specimen. 

XII.  Creatine. 

Folin-Benedict  and  Myers  Method.^ — To  20  c.c.  of  urine  in  a 
50  c.c.  volumetric  flask,  add  20  c.c.  of  normal  hydrochloric  acid  and 
place  the  flask  in  an  autoclave  at  a  temperature  of  117-120°  C.  for 
one-half  hour.  Add  distilled  water  until  the  volume  of  the  acid-urine 
mixture  is  exacdy  50  c.c,  close  the  flask  by  means  of  a  stopper,  and 
shake  it  thoroughly.  Approximately  neutralize  25  c.c.  of  this  mixture, 
introduce  it  into  a  500  c.c.  volumetric  flask  and  determine  its  creatinine 
content  according  to  Folin's  Method  (see  p.  415). 

Calculation. — Calculate  as  explained  on  p.  415,  and  from  this  value 
subtract  the  value  for  the  original  content  of  creatinine  before  hydrolysis. 
The  difference  between  these  two  values  will  be  the  creatine  content  of  the 
original  urine  in  terms  of  creatinine. 

' .  XIII.  Indican. 

Ellinger's  Method. — This  method  for  the  quantitative  determin- 
ation of  indican  is  based  upon  the  principle  underlying  Jaffe's  test  for 
the  detection  of  indican  (see  p.  298).  The  method  is  as  follows:  To 
50  c.c.  of  urine ^  in  a  small  beaker  or  casserole  add  5  c.c.  of  basic  lead 
acetate  solution,^  mix  thoroughly,  and  filter.  Transfer  40  c.c.  of  the 
filtrate  to  a  separatory  funnel,  add  an  equal  volume  of  Obermayer's 
reagent  (see  p.  299)  and  20  c.c.  of  chloroform,  and  extract  in  the  usual 

*  Benedict  and  Myers:   Am.  J .  Phys.,  18,  397,  1907. 

*  If  the  urine  under  examination  is  neutral  or  alkaline  in  reaction  it  should  be  made  faintly 
acid  with  acetic  acid  before  adding  the  basic  lead  acetate. 

^  For  preparation  of  basic  lead  acetate  solution  see  Appendix. 


urine:  quantitative  analysis.  417 

manner.  This  cxtnulion  with  chloroform  shcnild  he  repeated  until 
the  chloroform  solution  remains  colorless.  Shake  up  the  combined 
chloroform  extracts  2-3  times  with  distilled  water  in  a  separating  funnel 
and  complete  the  purification  by  extracting  with  very  dilute  sodium 
hydroxide  (1:1000).  Remove  all  traces  of  alkali  by  washing  with  water. 
Now  filter  the  combined  chloroform  extracts  through  a  dry  filter  pajjcr 
into  a  drv  Erlenmever  flask.  Distil  off  the  chloroform,  heat  the  residue 
on  a  boiling  water-bath  for  5  minutes  in  the  open  flask,  and  wash  the  dried 
residue  with  hot  water\  Add  10  c.c.  of  concentrated  sulphuric  acid  to 
the  washed  residue,  heat  on  the  water-bath  for  5-10  minutes,  dilute  with 
TOO  c.c.  of  water,  and  titrate  the  blue  solution  with  a  very  dilute  solution  of 
potassium  permanganate. ■  The  end-point  is  indicated  by  the  dissipation 
of  all  the  blue  color  from  the  solution  and  the  formation  of  a  pale  yellow 
color. 

Beautiful  plates  of  indigo  blue  sometimes  appear  in  the  chloroform 
extract  of  urines  containing  abundant  indican.  In  urines  preserved 
by  thymol  the  determination  of  indican  is  interfered  with  unless  great 
care  is  taken  in  washing  the  chloroform  extract  with  dilute  alkali.  Care 
should  be  taken,  therefore,  to  make  the  indican  determination  upon 
fresh  urine,  before  the  addition  of  the  preservative. 

Plasencia*  has  recently  suggested  a  method  which  is  shorter  than 
EUinger's  and  according  to  its  sponsor,  just  as  accurate. 

Calculation. — Ellinger  claims  that  one-sixth  of  the  amount  deter- 
mined must  be  added  to  the  value  obtained  by  titration  in  order  to  secure 
accurate  data.     This  correction  should  always  be  made. 

XIV.  Chlorides. 

Dehn-Clark  Method.^— In  this  method  the  organic  compounds, 
that  hold  the  chlorine  too  firmly  for  its  quantitative  precipitation  with 
silver  nitrate,  are  destroyed  by  oxidation  with  sodium  peroxide.  Sodium 
peroxide  in  the  presence  of  water  gives  off  nascent  oxygen  according  to  the 
following  equation:  * 

Na^O.  +  H,0— 2NaOH -^  O. 

The  oxygen  then  attacks  the  organic  matter  and  the  chlorine  is  left 
as  sodium  chloride.     The  procedure  is  as  follows:  To  10  c.c.  of  urine 

'  The  washing  should  be  continued  until  the  wash  water  is  no  longer  colored.  Ordi- 
narily two  or  three  washings  are  sufficient.  If  a  separation  of  indigo  particles  takes  place 
during  the  washing  process,  the  wash  water  should  be  filtered,  the  indigo  extracted  with  chloro- 
form, and  the  usual  method  applied  from  this  point. 

-A-" stock  solution"  of  potassium  permanganate  containing  3  grams  per  liter  should  be 
prepared,  and  when  needed  for  titration  purposes  a  suitable  volume  of  this  solution  should 
be  diluted  with  40  volumes  of  water.  The  potassium  permanganate  solution  should  be 
standardized  with  pure  indigo. 

'  Plasencia:   Revista  de  Medicina  y  Cirugia.,  17,  i,  191 2. 

'Private  communication  to  the  author  from  Mr.  S.  C.  Clark. 

27 


4l8  PHYSIOLOGICAL    CHEMISTRY. 

in  a  75-100  c.c.  casserole,  add  i. 0-1.2  gram  of  sodium  peroxide  and 
evaporate  the  mixture  to  dryness  on  a  boiling  water-bath.  In  case  the 
residue  is  not  pure  white,  thus  indicating  that  insufficient  sodium  peroxide 
has  been  added,  the  residue  should  be  moistened  with  distilled  water, 
additional  sodium  peroxide  added,  and  the  mixture  again  evaporated  to 
dryness.  When  the  oxidation  is  complete,  treat  the  mass  with  10-20  c.c. 
of  distilled  water  and  stir  until  it  has  practically  all  been  brought  into 
solution.  Then  introduce  a  bit  of  litmus  paper  and  add  dilute  nitric  acid 
(1:1)  until  the  litmus  paper  turns  red  and  all  effervescence  ceases.  Now 
place  the  casserole  on  a  hot  plate  or  on  a  gauze  and  heat  the  contents 
almost  to  the  boiling-point.^  To  the  hot  solution  add  a  standard  solution 
of  silver  nitrate  (see  page  419)  in  slight  excess.^  Filter  off  the  silver 
chloride  while  the  solution  is  still  hot  and  wash  the  precipitate  thoroughly 
with  distilled  water.  To  the  filtrate,  add  i  c.c.  of  a  saturated  solution  of 
ferric  ammonium  sulphate  and  then  titrate  with  a  standard  solution  of 
ammonium  thiocyanate  (see  page  420)  until  the  clear,  slightly  yellow 
fluid  (or  the  opalescent,  milky  fluid,  in  case  there  is  much  excess  of  silver 
nitrate)  changes  to  a  slight  reddish-brown  color.  The  color  of  the  end- 
point  varies  with  the  individual.  The  exact  end-point  reached  is  not  so 
important  as  is  the  securing  of  the  same  end-point  in  a  series  of  deter- 
minations as  that  obtained  in  the  standardization  of  the  standard  solutions 
used. 

Calculation.- — The  standard  solution  of  silver  nitrate  should  be 
made  up  so  that  i  c.c.  equals  o.oio  gram  of  sodium  chloride  and  i  c.c. 
of  the  ammonium  thiocyanate  should  be  equivalent  to  i  c.c.  of  the  silver 
nitrate  solution  (see  p.  419).  Then,  if  the  number  of  cubic  centimeters 
of  ammonium  thiocyanate  used  be  subtracted  from  the  number  of  cubic 
centimeters  of  silver  nitrate,  the  difference  is  the  number  of  cubic  centi- 
meters of  silver  nitrate  actually  used  in  the  precipitation  of  chlorine  as 
silver  chloride.  This  number,  multiplied  by  0.0 10,  gives  the  weight  in 
grams  of  the  sodium  chloride  in  the  10  c.c.  of  urine  used.  If  it  is  desired  to 
express  the  result  in  percentage  of  sodium  chloride,  move  the  decimal 
point  one  place  to  the  right. 

In  a  similar  manner  the  weight  or  percentage  of  chlorine  may  be  com- 
puted, using  the  factor  0.006  as  explained  in  Mohr's  method,  below. 
Calculate  the  quantity  of  sodium  chloride  and  of  chlorine  in  the  twenty- 
four-hour  urine  specimen. 

2.  Mohr's  Method. — To  10  c.c.  of  urine  in  a  small  platinum  or  porce- 

'  If  there  is  a  slight  precipitate,  due  to  silicic  acid  from  the  casserole,  this  is  filtered  off 
and  the  filtrate  collected  in  a  200  c.c.  beaker. 

^  This  point  is  most  easily  recognized  by  keeping  the  solution  hot  and  in  constant  agitation 
while  adding  the  silver  nitrate  so  that  the  silver  chlori  le  formed  coagulates  and  sinks,  leaving 
a  clear,  supernatant  fluid. 


urine:  quantitative  analysis.  419 

lain  crucible  or  dish  add  about  2  grams  of  chlorine-free  potassium  nitrate 
and  evaporate  to  dryness  at  100°  C.  (The  evaporation  may  be  con- 
ducted over  a  low  tlame  provided  care  is  taken  to  prevent  loss  by  sj)urting.) 
By  means  of  crucible  tongs  hold  the  crucible  or  dish  over  a  free  flame  until 
all  carbonaceous  matter  has  disappeared  and  the  fused  mass  is  slightly 
yellow  in  color.  Cool  the  residue  somewhat  and  bring  it  into  solution  in 
a  small  amount  (15-25  c.c.)  of  distilled  water  acidified  with  about  10  drops 
of  nitric  acid.  Transfer  the  solution  to  a  small  beaker,  being  sure  to 
rinse  out  the  crucible  or  dish  very  carefully.  Test  the  reaction  of  the 
fluid,  and  if  not  already  acid  in  reaction  to  litmus,  render  it  slightly  acid 
with  nitric  acid.  Now  neutralize  the  solution  by  the  addition  of  calcium 
carbonate'  in  substance,  add  2-5  drops  of  neutral  potassium  chromate 
solution  to  the  mixture,  and  titrate  with  a  standard  silver  nitrate  solution.^ 

This  standard  solution  should  be  run  in  from  a  burette,  stirring  the 
liquid  in  the  beaker  after  each  addition.  The  end-reaction  is  reached 
when  the  yellow  color  of  the  solution  changes  to  a  slight  orange-red.  At 
this  point  take  the  burette  reading  and  compute  the  percentage  of  chlor- 
ine and  sodium  chloride  in  the  urine  examined. 

Calculation. — Since  i  c.c.  of  the  standard  silver  nitrate  solution  is 
equivalent  to  0.0 10  gram  of  sodium  chloride,  to  obtain  iheweight,  in  grams, 
of  the  sodium  chloride  in  the  10  c.c.  of  urine  used  multiply  the  number  of 
cubic  centimeters  of  standard  solution  used  by  o.oio.  If  it  is  desired  to 
express  the  result  in  percentage  of  sodium  chloride  move  the  decimal  point 
one  place  to  the  right. 

To  obtain  the  weight,  in  grams,  of  the  chlorine  in  the  10  c.c.  of  urine 
used  multiply  the  number  of  cubic  centimeters  of  standard  solution  used 
by  0.006,  and  if  it  is  desired  to  express  the  result  in  percentage  of  chlorine 
move  the  decimal  point  one  place  to  the  right. 

Calculate  the  quantity  of  sodium  chloride  and  chlorine  in  the  twenty- 
four-hour  urine  specimen. 

3.  Volhard-Arnold  Method. — Place  10  c.c.  of  urine  in  a  100  c.c. 
volumetric  flask,  add  20-30  drops  of  nitric  acid  (sp.  gr.  i .  2)  and  2  c.c.  of 
a  cold  saturated  solution  of  ferric  alum.  If  necessary,  at  this  point  a  few 
drops  of  an  8  per  cent  solution  of  potassium  permanganate  may  be  added 
to  dissipate  the  red  color.  Now  slowly  run  in  a  known  volume  of  the 
standard  silver  nitrate'  solution  (20  c.c.  is  ordinarily  used)  in  order  to 
precipitate  the  chlorine  and  insure  the  presence  of  a.n  excess  oi  silver  nitrate. 
The  mixture  should  be  continually  shaken  during  the  addition  of  the 

'  The  cessation  of  eflervescence  and  the  presence  of  some  undecomposed  calcium  car- 
bonate at  the  bottom  of  the  vessel  are  the  indications  of  neutralization. 

'  Standard  silver  nitrate  solution  may  be  prepared  by  dissolving  29.042  grams  of  silver 
nitrate  in  i  liter  of  distilled  water.  Each  cubic  centimeter  of  this  solution  is  equivalent  to 
O.OIO  gram  of  sodium  chloride  or  to  0.006  gram  of  chlorine. 


420  PHYSIOLOGICAL    CHEMISTRY. 

standard  solution.  Allow  the  flask  to  stand  lo  minutes,  then  fill  it  to  the 
loo  ex.  graduation  with  distilled  water  and  tlioroughly  mix  the  contents. 
Now  filter  the  mixture  through  a  dry  filter  paper,  collect  50  c.c.  of  the 
filtrate  and  titrate  it  with  standardized  ammonium  thiocyanate  solution.^ 
The  first  permanent  tinge  of  red-brown  indicates  the  end-point.  Take 
the  burette  reading  and  compute  the  weight  of  sodium  chloride  in  the 
10  c.c.  of  urine  used. 

Calculation. — The  number  of  cubic  centimeters  of  ammonium  thio- 
cyanate solution  used  indicates  the  excess  of  standard  silver  nitrate 
solution  in  the  50  c.c.  of  filtrate  titrated.  Multiply  this  reading  by  2,  in- 
asmuch as  only  one-half  of  the  filtrate  was  employed,  and  subtract  this 
product  from  the  number  of  cubic  centimeters  of  silver  nitrate  (20  c.c.) 
originally  used,  in  order  to  obtain  the  actual  number  of  cubic  centimeters 
of  silver  nitrate  utilized  in  the  precipitation  of  the  chlorides  in  the  10  c.c. 
of  urine  employed. 

To  obtain  the  weight  in  grams  of  the  sodium  chloride  in  the  10  c.c. 
of  urine  used,  multiply  the  number  of  cubic  centimeters  of  the  standard 
silver  nitrate  solution,  actually  utilized  in  the  precipitation,  by  o.oio. 
If  it  is  desired  to  express  the  result  in  percentage  of  sodium  chloride  move 
the  decimal  point  one  place  to  the  right. 

In  a  similar  manner  the  weight,  or  percentage  of  chlorine  may  be  com- 
puted using  the  factor  0.006  as  explained  in  Mohr's  method,  page  418. 

Calculate  the  quantity  of  sodium  chloride  and.  chlorine  in  the  twenty- 
four-hour  urine  specimen. 

4,  Volhard-Harvey  Method.- — Introduce  5  c.c.  of  urine  into  a 
small  porcelain  evaporating  dish  or  casserole  and  dilute  with  about  20  c.c. 
of  distilled  water.  Precipitate  the  chlorides  by  the  addition  of  10  c.c.  of 
standard  silver  nitrate  solution^  and  add  2  c.c.  of  acidified  indicator.* 
Now  run  in  a  standard  ammonium  thiocyanate  solution^  from  a  burette 

*  This  solution  is  made  of  such  a  strength  that  i  c.c.  of  it  is  equal  to  i  c.c.  of  the  standard 
silver  nitrate  solution  used.  To  prepare  the  solution  dissolve  13  grams  of  ammonium 
thiocyanate,  NH^SCN,  in  a  little  less  than  a  liter  of  water.  In  a  small  flask  place  20  c.c. 
of  the  standard  silver  nitrate  solution,  5  c.c.  of  the  ferric  alum  solution  and  4  c.c.  of  nitric 
acid  (sp.  gr.  1.2),  add  water  to  make  the  total  volume  100  c.c.  and  thoroughly  mix  the  contents 
of  the  flask.  Now  run  in  the  ammonium  thiocyanate  solution  from  a  burette  until  a  per- 
manent red-brown  tinge  is  produced.  This  is  the  end-reaction  and  indicates  that  the  last 
trace  of  silver  nitrate  has  been  precipitated.  Take  the  burette  reading  and  calculate  the 
amount  of  water  necessary  to  use  in  diluting  the  ammonium  thiocyanate  in  order  that  10  c.c. 
of  this  solution  may  be  exactly  equal  to  10  c.c.  of  the  silver  nitrate  solution.  Make  this  dilution 
and  titrate  again  to  be  certain  that  the  solution  is  of  the  proper  strength. 

^  Harvey:    Archives  of  Internal  Medicine,  6,  12,  1910, 
^  See  p.  419. 

*  This  is  prepared  as  follows:  To  30  c.c.  of  distilled  water  add  70  c.c.  of  33  per  cent 
nitric  acid  (sp.  gr.  1.2)  and  dissolve  100  grams  of  crystalline  ferric  ammonium  sulphate  in  this 
dilute  acid  solution.  Filter  and  use  the  filtrate  which  is  a  saturated  solution  of  the  iron  salt. 
This  single  reagent  takes  the  place  of  the  nitric  acid  and  ferric  alum  as  used  in  Volhard- 
Arnold  method,  and  insures  the  use  of  the  proper  quantity  of  acid. 

*  This  is  a  solution  of  ammonium  thiocyanate  of  such  a  strength  that  2  c.c.  is  equivalent 
to  I  c.c.  of  the  silver  nitrate  solution.     First  make  a  concentrated  solution  by  dissolving   13 


urine:  quantitative  analysis.  421 

until  a  faint  red-brown  tint  is  visible  throughout  the  mixture.  This 
point  may  be  determined  readily  by  permitting  the  precipitate  to  settle 
somewhat.  Calculate  the  sodium  chloride  value  so  indicated  below. 
(If  a  red  tint  is  produced  when  the  first  drop  of  thiocyanate  is  added 
an  additional  to  c.c.  of  the  standard  silver  nitrate  solution  must  be  in- 
troduced. The  titration  should  then  proceed  as  above  described  and 
proper  allo^'ance  made  in  the  calculation  for  the  extra  volume  of  silver 
nitrate  employed.) 

Calculation. — Since  2  c.c.  of  the  ammonium  thiocyanate  solution  is 
equivalent  to  i  c.c.  of  the  silver  nitrate  solution,  divide  the  burette  reading 
by  2  and  subtract  the  quotient  from  10  c.c,  the  quantity  of  silver  nitrate 
solution  taken.  This  value  is  the  number  of  cubic  centimeters  of  silver 
nitrate  solution  actually  used  in  the  precipitation  of  the  chlorides.     As 

1  c.c.  of  the  silver  nitrate  solution  is  equivalent  to  o.oi  gram  of  sodium 
chloride,  the  number  of  cubic  centimeters  of  silver  nitrate  solution  used 
multiplied  by  o.oi  gram  will  give  the  weight  of  sodium  chloride  in  the  5 
c.c.  portion  of  urine  used.  The  weight  of  chlorine  may  be  computed  by 
using  the  factor  0.006  as  explained  under  Mohr's  method,  page  418. 

Calculate  the  weight  of  sodium  chloride  and  chlorine  in  the  twenty- 
four-hour  urine  specimen. 

A  "short  cut"  method  of  calculating  the  twenty-four-hour  output  of 
sodium  chloride  consists  in  subtracting  the  burette  reading  from  20  c.c, 
multiplying  this  value  by  the  total  urine  volume  and  pointing  off  three 
places. 

XV.  Acetone  and  Diacetic  Acid. 

I.  Folin-Hart  Method. ^This  method  serves  the  same  purpose  as 
the  Messinger-Huppert  Method,  i.  e.,  the  determination  of  both  acetone 
and  diacetic  acid  in  terms  of  acetone.  It  is,  however,  much  simpler  and 
less  time-consuming.  The  method  includes  the  transformation  of  the 
diacetic  acid  into  acetone  and  carbon  dioxide  by  means  of  heat  and  the 
subsequent  removal  of  the  acetone  thus  formed,  as  well  as  the  preformed 
acetone,  by  means  of  an  air  current  as  first  suggested  by  Folin  (see  p. 
399).     The  procedure  is  as  follows:  Introduce  into  a  wide-mouthed  bottle 

grams  in  one  liter  of  water.     To  determine  the  requisite  dilution  to  make  such  a  solution  that 

2  c.c.  shall  be  equivalent  to  i  c.c.  of  the  silver  nitrate  solution  proceed  as  follows:  Introduce 
10  c.c.  of  the  silver  nitrate  solution  into  a  small  porcelain  evaporating  .dish  or  casserole,  add 
30-50  c.c.  of  distilled  water,  2  c.c.  of  the  acid  indicator  and  titrate  as  described  above  with 
the  ammonium  thiocyanate  solution.  The  total  volume  of  the  concentrated  thiocyanate 
solution  including  that  used  in  this  titration  is  divided  by  ten,  and  the  result  multiplied  by  the 
difference  between  this  burette  reading  and  20  c.c.  This  will  give  the  volume  of  distilled  water 
which  must  be  added  to  the  concentrated  thiocyanate  solution  to  render  2  c.c.  equivalent  to 
I  c.c.  of  the  silver  nitrate  solution. 


422 


PHYSIOLOGICAL    CHEMISTRY. 


2CO  c.c.  of  water,  an  accurately  measured  excess  of  N/io  iodine  solution^ 
and  an  excess  of  40  per  cent  potassium  hydroxide.  Prepare  an  aerometer 
cylinder  containing  alkaline  hypoiodite  solution  to  absorb  any  acetone 
which  may  be  present  in  the  air  of  the  laboratory,  and  between  the  cylinder 
and  bottle  suspend  a  test-tube  about  two  inches  in  diameter.  This 
large  test-tube  should  contain  20  c.c.  of  the  urine  under  examination,  10 
drops  of  a  10  per  cent  solution  of  phosphoric  acid,  10  grams  of  sodium 
chloride,  and  a  little  petroleum,  and  should  be  raised  sufficiently  high  to 
facilitate  the  easy  application  of  heat  to  its  bottom  portion.  The  con- 
nections on  the  side  of  the  tube  should  be  provided  with  bulb-tubes 
containing  cotton.  When  the  apparatus  is  arranged  as  described,  it 
should  be  connected  with  a  Chapman  pump  and  an  air  current  passed 
through  for  twenty-five  minutes.  During  this  period  the  contents  of 
the  test-tube  are  heated  just  to  the  boiling-point  and  after  an  interval  of 
five  minutes  again  heated  in  the  same  manner.  By  this  means  the 
diacetic  acid  is  converted  into  acetone  and  at  the  end  of  the  twenty-five- 
minute  period  this  acetone,  as  well  as  the  preformed  acetone,  will  have 
been  removed  from  the  urine  to  the  absorption  bottle  and  there  retained 
as  iodoform. 

The  contents  of  the  absorption  bottle  should  now  be  acidified  with 
concentrated  hydrochloric  acid,^  and  titrated  with  N/io  sodium  thio- 
sulphate  and  starch  as  in  the  Messinger-Huppert  method  (see  below). 

2.  Messinger-Huppert  Method.^ — ^Place  100  c.c.  of  urine  in  a  dis- 
tillation flask  and  add  2  c.c.  of  50  per  cent  acetic  acid.  Connect  the 
flask  with  a  condenser,  properly  arrange  a  receiver,  attach  a  terminal 
series  of  bulbs  containing  water,  and  distil  over  about  nine-tenths  of  the 
urine  mixture.     Remove  the  receiver,  attach  another,  and  subject  the  resid- 

'  Proceed  as  follows  in  order  to  obtain  a  rough  idea  regarding  the  amount  of  N/io  iodine 
solution  to  be  used:  Introduce  into  a  test-tube  lo  c.c.  of  the  urine  under  examination  and 
I  c.c.  of  a  solution  of  ferric  chloride  made  by  dissolving  loo  grams  of  ferric  chloride  in  loo  c.c. 
of  distilled  water.  After  permitting  the  mixture  to  stand  for  two  minutes,  compare  the 
color  with  that  of  an  equal  volume  of  the  ferric  chloride  solution  in  a  test-tube  of  similar 
diameter.  If  the  two  solutions  be  of  approximately  the  same  color  intensity,  20  c.c.  of  the 
urine  under  examination  will  yield  sufficient  acetone  to  require  nearly  10  c.c.  of  N/io  iodine 
solution.  In  case  the  mixture  is  darker  in  color  than  is  the  ferric  chloride  solution,  the  former 
should  be  diluted  with  distilled  water  until  it  it  of  approximately  the  same  intensity  as  the 
ferric  chloride  solution.  From  this  data  the  amount  of  N/io  iodine  solution  required  may  be 
roughly  estimated  by  means  of  the  following  table: 


Urine  c.c. 

Ferric  chloride. 

10 

1 

ID 

I 

10 

I 

10 

I 

Water. 


N/io  Iodine  required  c.c. 


10 

10 

20 

20 

35 

30 

50 

*  An  excess  of  iodine  is  indicated  by  the  development  of  a  brown  color 

^  This  method  serves  to  determine  bolk  acetone  and  diacetic  acid  in  terms  of  acetone. 


urine:  quantitativk  analysis.  423 

ual  portion  of  the  mixture  to  a  second  distillation.  Test  this  fluid  for 
acetone  and  if  the  presence  of  acetone  is  indicated  add  about  roo  c.c.  of 
water  to  the  residue  and  again  distil.  Treat  the  united  acetone  distillates 
with  I  c.c.  of  dilute  (12  per  cent)  sulphuric  acid  and  redistil,  collecting 
this  second  distillate  in  a  glass-stoppered  flask.  During  distillation,  how- 
ever, the  glass  stopper  is  replaced  by  a  cork  with  a  double  perforation,  the 
glass  tube  from  one  perforation  passing  to  the  condenser,  while  the 
bulbs  containing  water,  before  mentioned,  are  attached  by  means  of  the 
tube  in  the  other  perforation.  Allow  the  distillation  process  to  proceed 
until  practically  all  of  the  fluid  has  passed  over,  then  remove  the  receiving  . 
flask  and  insert  the  glass  stopper.  Now  treat  the  distillate  carefully 
with  10  c.c.  of  a  N/io  solution  of  iodine  and  add  sodium  hydroxide 
solution,  drop  by  drop,  until  the  blue  color  is  dissipated  and  the  iodoform 
precipitates.  Stopper  the  flask  and  shake  it  for  about  one  minute, 
acidify  the  solution  with  concentrated  hydrochloric  acid,  and  note  the 
production  of  a  brown  color  if  an  excess  of  iodine  is  present.  In  case 
there  is  no  such  excess,  the  solution  should  be  treated  with  N/io  iodine 
solution  until  an  excess  is  obtained.  Retitrate  this  excess  of  iodine  with 
N/io  sodium  thiosulphate  solution  until  a  light  yellow  color  is  observed. 
At  this  point  a  few  cubic  centimeters  of  starch  paste  should  be  added  and 
the  mixture  again  titrated  until  no  blue  color  is  ^'isible.  This  is  the  end- 
reaction. 

Calculatim. — Subtract  the  number  of  cubic  centimeters  of  N/io 
thiosulphate  solution  used  from  the  volume  of  N/io  iodine  solution 
employed.  Since  i  c.c.  of  the  iodine  solution  is  equivalent  to  0.967 
milligram  of  acetone,  and  since  i  c.c.  of  the  thiosulphate  solution  is 
equivalent  to  i  c.c.  of  the  iodine  solution,  if  we  multiply  the  remainder 
from  the  above  subtraction  by  0.967  we  will  obtain  the  number  of 
milligrams  of  acetone  in  the  100  c.c.  of  urine  examined. 

Calculate  the  quantity  of  acetone  in  the  twenty-four-hour  urine 
specimen. 

XVI.  Acetone. 

I.  Folin's  Method. — The  same  type  of  apparatus  is  used  in  this 
method  as  that  described  in  Folin's  method  for  the  determination  of 
ammonia  (see  p.  399).  The  procedure  is  as  follows:  Introduce  20-25 
c.c.  of  the  urine  under  examination  into  the  aerometer  cylinder  and 
add  10  drops  of  10  per  cent  phosphoric  acid,^  8-10  grams  of  sodium 
chloride,^  and  a  little  petroleum.     Introduce  into  an  absorption  flask, ^ 

'  Oxalic  acid  (0.2  —  0.3  gram)  may  be  substituted  if  desired. 

-  Acetone  is  insoluble  in  a  saturated  solution  of  sodium  chloride. 

^Folin's  improved  absorption  tube  (see  Fig.  128,  p.  400)  should  be  used  in  this  connec- 
tion inasmuch  as  the  original  type  embracing  the  use  of  a  rubber  stopper  is  unsatisfactory  be- 
cause of  the  solvent  action  of  alkaline  hypoiodite  on  rubber. 


424  PHYSIOLOGICAL    CHEMISTRY. 

such  as  is  used  in  the  ammonia  determination  (see  p.  399),  150  c.c.  of 
water,  10  c.c.  of  a  40  per  cent  solution  of  potassium  hydroxide,  and  an 
excess  of  a  N/io  iodine  solution.  Connect  the  flask  with  the  aerometer 
cylinder,  attach  a  Chapman  pump,  and  permit  an  air  current,  slightly 
less  rapid  than  that  used  for  the  determination  of  ammonia,  to  be  drawn 
through  the  solution  for  20-25  minutes.  All  of  the  acetone  will,  at  this 
point,  have  been  converted  into  iodoform  in  the  absorption  flask.  Add 
10  c.c.  of  concentrated  hydrochloric  acid  (a  volume  equivalent  to  that  of 
the  strong  alkali  originally  added),  to  the  contents  of  the  latter  and 
titrate  the  excess  of  iodine  by  means  of  N/io  sodium  thiosulphate  solution 
and  starch,  as  in  the  Messinger-Huppert  method  (see  p.  422), 

Folin  has  further  made  suggestions  regarding  the  simultaneous  deter- 
mination of  acetone  and  ammonia  by  the  use  of  the  same  air  current.^ 
This  is  an  important  consideration  for  the  clinician  inasmuch  as  urines 
which  contain  acetone  and  diacetic  acid  are  generally  those  from  which 
the  ammonia  data  are  also  desired.  The  procedure  for  the  combination 
method  is  as  follows:  Arrange  the  ammonia  apparatus  as  usual  (see  p. 
399),  and  to  the  aerometer  of  the  ammonia  apparatus  attach  the  acetone 
apparatus  set  up  as  described  above.  Regulate  the  air  current  with 
special  reference  to  the  determination  of  acetone  and  at  the  end  of  20-25 
minutes  disconnect  the  acetone  apparatus  and  complete  the  determination 
of  the  acetone  as  just  described.  The  air  current  is  not  interrupted,  and 
after  having  run  one  and  one-half  hours  the  ammonia  apparatus  is  de- 
tached and  the  ammonia  determination  completed  as  described  on  page 

399- 

If  data  regarding  diacetic  acid  are  desired,  the  result  obtained  by 
Folin's  method  may  be  subtracted  from  the  result  obtained  by  the  Mes- 
singer-Huppert method  (see  p.  422),  inasmuch  as  the  latter  method 
determines  both  acetone  and  diacetic  acid.  Under  all  conditions  the 
determination  of  acetone  should  be  as  expeditious  as  possible.  This 
is  essential,  not  only  because  of  the  fact  that  any  diacetic  acid  present 
in  the  urine  will  become  transformed  into  acetone,  but  also  because  of  the 
rapid  spontaneous  decomposition  of  the  alkaline  hypoiodite  solution  used 
in  the  determination  of  the  acetone.  It  has  been  claimed  that  alkaline 
hypoiodite  solutions  are  almost  completely  converted  into  iodate  solutions 
in  one-halj  hour.  Folin  states,  however,  that  the  transformation  is  not 
so  rapid  as  this,  but  he  nevertheless  emphasizes  the  necessity  of  rapidity 
of  manipulation.  At  the  same  time  it  should  be  remembered  that  the 
air  current  must  not  be  as  rapid  as  for  ammonia,  inasmuch  as  the  alkaline 
hypoiodite  solution  will  not  absorb  all  the  acetone  under  those  conditions. 

'  These  determinations  may  even  be  made  on  the  same  sample  of  urine  if  the  sample  is 
too  small  for  the  double  determination.  - 


urine:  quantitative  analysis.  425 

XVII.  Diacetic  Acid. 

1.  Folin-Hart  Method. — Arrange  the  ap])aratus  as  described  under 
the  Folin-Hart  method  for  the  determination  of  acetone  and  diacetic  acid 
(see  p.  421).  Start  the  air  current  in  the  usual  way  and  permit  it  to  run 
25  minutes  •without  the  application  of  heat  to  the  urine  under  examination. 
Under  these  conditions  the  preformed  acetone  present  in  the  solution  is 
all  removed  (^ee  p.  423).  Immediately  attach  a  freshly  prepared  absorp- 
tion bottle  or  introduce  fresh  alkaline  hypoiodite  solution  into  the  original 
bottle.  Apply  heat  to  the  large  test-tube  as  already  described  (see  p.  422), 
in  order  to  convert  the  diacetic  acid  into  acetone,  permit  the  air  current  to 
continue  for  the  usual  25-minute  period,  and  determine  the  diacetic  acid 
value  in  terms  of  acetone  by  the  usual  titration  procedure  (see  p.  422). 

2.  Folin-Messinger-Huppert  Method. — Determine  the  combined 
acetone  and  diacetic  acid,  in  terms  of  acetone,  by  the  Messinger-Huppert 
method  (see  p.  422),  and  subsequently  determine  the  acetone  by  Folin's 
method  (see  p.  423) ,  Subtract  the  value  determined  by  the  second  method 
from  that  obtained  in  the  first  method  to  secure  data  regarding  the  diacetic 
acid  content  of  the  urine,  in  terms  of  acetone. 

XVIII.  ^5-Oxybutyric  Acid. 

I.  Shaffer's  Method.— Introduce  25-250  c.c.  of  urine ^  into  a  500 
c.c.  volumetric  flask  and  add  an  excess  of  basic  lead  acetate  and  10  c.c. 
of  concentrated  ammonium  hydroxide.  Dilute  the  mixture  to  the  500 
c.c.  mark,  shake  the  flask  thoroughly  and  filter.  Transfer  200  c.c.  of 
the  filtrate  to  an  800  c.c.  Kjeldahl  distilling  flask,  add  300-400  c.c.  of 
water,  15  c.c.  of  concentrated  sulphuric  acid  and  a  little  talcum  and 
distil  the  mixture  until  200  to  250  c.c.  of  distillate  has  been  collected  (A).^ 
To  this  distillate  (A),  which  contains  acetone  (both  preformed  and  that 
produced  from  diacetic  acid),  3^x16.  volatile  fatty  acids  is  added  5  c.c.  of  10 
percent  potassium  hydroxide  and  the  distillate  redistilled  in  order  to 
remove  the  volatile  fatty  acids. ^  This  second  distillate  (A,)  is  then 
titrated  with  standard  iodine  and  thiosulphate  (see  p.  423).  The  urine- 
sulphuric  acid  residue  from  which  distillate  A  was  obtained  is  again 

'  The  amount  used  depends  upon  the  expected  yield  of  ^-o.xj-butyric  acid.  In  the  case 
of  urines  which  give  a  strong  ferric  chloride  reaction  for  diacetic  acid,  or  when  5-10  grams 
or  more  of  /J-oxybutyric  acid  is  expected,  it  is  unnecessary-  to  use  more  than  25-50  c.c.  of 
urine.  However,  in  case  only  a  trace  of  /^-o-xybutyric  acid  is  expected,  the  volume  should  be 
much  larger  as  indicated.  Under  all  conditions,  the  amount  specified  is  sufficient  for  duplicate 
determinations.  It  is  desirable  to  use  such  a  volume  of  urine  as  contains  the  proper  amount  of 
/9-oxybutyric  acid  to  yield  25-50  milligrams  of  acetone. 

*  This  distilling  flask  should  be  provided  with  a  dropping  tube,  by  means  of  which  water 
may  be  introduced  in  order  to  prevent  the  contents  of  the  flask  from  becoming  less  than 
400  c.c.  in  volume.  Care  should  be  taken  to  use  a  good  condenser  in  the  distillation,  but  it 
is  not  necessary  to  cool  the  distillate  with  ice. 

^  Formic  acid  is  one  of  the  most  troublesome. 


426  PHYSIOLOGICAL   CHEMISTRY. 

distilled,  400-600  c.c.  of  a  0.1-0.5  P*^^  cent  potassium  bichromate  solu- 
tion, being  added,  by  means  of  the  dropping  tube,  during  the  process  of 
distillation.^  In  adding  the  bichromate,  care  should  be  taken  not  to 
add  it  faster  than  the  distillate  collects  except  in  cases  where  the  boiling 
fluid  assumes  a  pure  green  color,  thus  indicating  that  the  bichromate  is 
being  used  up  more  rapidly.  After  about  500  c.c.  of  distillate  (B)  has 
collected,  20  c.c.  of  a  3  per  cent  solution  of  hydrogen  peroxide  and  a 
few  cubic  centimeters  of  potassium  hydroxide  solution  are  added  and 
the  mixture  (B)  subjected  to  redistillation.  Distil  off  about  300  c.c.  and 
titrate  this  distillate  (B2)  as  usual  with  iodine  and  thiosulphate  (see  p.  423.) 
Calculation. — The  author  advises  the  use  of  solutions  of  thiosulphate 
and  iodine,  which  are  a  trifle  stronger  than  N/io;  i.  e.,  103.  4  N/io. 
Each  cubic  centimeter  of  an  iodine  solution  of  this  strength  is  equivalent 
to  one  milligram  of  acetone  or  to  1.794  milligrams  of  /?-oxybutyric  acid. 
The  thiosulphate  solution  is  accepted  as  the  standard  and  should  be 
restandardized,  from  time  to  time,  by  a  N/io  solution  of  potassium 
bi-iodate. 

2.  Black's  Method. — Render  50  c.c.  of  the  urine  under  examination, 
faintly  alkaline  with  sodium  carbonate  and  evaporate  to  one-third  the 
original  volume.  Concentrate  to  about  10  c.c.  on  a  water-bath,  cool  the 
residue,  acidify  it  with  a  few  drops  of  concentrated  hydrochloric  acid^ 
and  add  plaster  of  Paris  to  form  a  thick  paste.  Permit  the  mixture  to 
stand  until  it  begins  to  "set,"  then  break  it  up  with  a  stout  glass  rod 
having  a  blunt  end  and  reduce  the  material  to  the  consistency  of  a  fairly 
dry  coarse  meal.^  Transfer  the  meal  to  a  Soxhlet  apparatus  and  extract 
with  ether  for  two  hours.  At  the  end  of  this  period  evaporate  the  ether- 
extract  either  spontaneously  or  in  an  air  current.  Dissolve  the  residue 
in  water,  add  a  little  bone-black,  if  necessary,  filter  until  a  clear  solution 
is  obtained  and  make  up  the  filtrate  to  a  known  volume  (25  c.c.  or  less) 
with  water.  The  /?-oxybutyric  acid  should  then  be  determined  by  means 
of  the  polariscope. 

3.  Darmstadter's  Method. — This  method  is  based  on  the  fact  that 
crotonic  acid  is  formed  from  /5-oxybutyric  acid  under  the  influence  of 
concentrated  mineral  acids.  The  method  is  as  follows:  Render  100  c.c. 
of  urine  slightly  alkaline  with  sodium  carbonate  and  evaporate  nearly  to 
dryness  on  a  water-bath.  Dissolve  the  -esidue  in  150-200  c.c.  of  50-55 
per  cent  sulphuric  acid,  transfer  the  acid  solution  to  a  i-liter  distillation 
flask  and  connect  it  with  a  condenser.     Through  the  cork  of  the  flask 

'  Generally  the  addition  of  0.5  gram  of  potassium  bichromate  is  sufficient.  In  case 
the  urine  contains  a  high  concentration  of  sugar  or  when  a  large  volume  of  urine  is  used, 
it  may  be  necessary  to  use  2-3  grams  of  the  bichromate. 

^  The  residue  should  give  a  distinct  red  color  with  litmus  paper. 

*  Before  this  is  accomplished  it  may,  in  some  cases,  be  necessary  to  add  a  little  more 
plaster  of  Paris. 


urine:  quantitative  analysis.  427 

introduce  the  stem  of  a  dropping  funnel  containing  water.  Heat  the 
flask  gently  until  foaming  ceases,  then  use  a  full  flame  and  distil  over 
about  300-350  c.c.  of  fluid,  keeping  the  volume  of  litjuid  in  the  distillation 
flask  constant  by  the  addition  of  water  from  the  dropping  funnel  as  the 
distillate  collects.  Ordinarily  it  will  take  about  2-2  1/2  hours  to  collect 
this  amount  of  distillate.  Extract  the  distillate  three  times ^  with  ether 
in  a  separatopy  funnel,  evaporate  the  ether  and  heat  the  residue  at  160° 
C.  for  a  few  minutes  to  remove  volatile  fatty  acids.  Dissolve  the  residue 
in  50  c.c.  of  water,  filter  and  titrate  this  aqueous  solution  of  crotonic 
acid  with  N/io  sodium  hydroxide  solution,  using  phenolphthalcin  as 
indicator. 

Calciilatimi. — One  c.c.  of  N/io  sodium  hydroxide  solution  equals 
0.0086  gram  of  crotonic  acid,  i  part  of  crotonic  acid  equals  1.2 1  part  of 
/?-oxybutyric  acid,  and  i  c.c.  of  N/io  sodium  hydroxide  solution  equals 
0.0 104 1  gram  of  ;9-oxybutyric  acid.  To  compute  the  quantity  of  fi- 
oxybutyric  acid,  in  grams,  multiply  the  number  of  cubic  centimeters  of 
N/io  sodium  hydroxide  solution  used  by  0.01041. 

4.  Bergell's  Method. — Render  100-300  c.c.  of  sugar-free'  urine 
slightly  alkaline  with  sodium  carbonate,  evaporate  the  alkaline  urine 
to  a  syrup  on  a  water-bath,  cool  the  syrup,  rub  it  up  with  syrupy  phos- 
phoric acid  (being  careful  to  keep  the  mixture  cool),  20-30  grams  of 
finely  pulverized,  anhydrous  copper  sulphate,  and  20-25  grams  of  flne 
sand.  Mix  the  mass  thoroughly,  place  it  in  a  paper  extraction  thimble^ 
and  extract  the  dry  mixture  with  ether  in  a  Soxhlet  apparatus  (Fig.  136, 
page  437).  Evaporate  the  ether,  dissolve  the  residue  in  about  25  c.c.  of 
water,  decolorize  the  fluid  with  animal  charcoal,  if  necessary,  and  deter- 
mine the  content  of  .-^-oxybutyric  acid  by  a  polarization  test. 

5.  Boekelman  and  Bouma's  Method. — Place  25  c.c.  of  urine  in 
a  flask,  add  25  c.c.  of  12  per  cent  sodium  hydroxide  and  25  c.c.  of  benzoyl 
chloride,  stopper  the  flask  and  shake  it  vigorously  for  three  minutes 
under  cold  water.  Remove  the  clear  fluid  by  means  of  a  pipette,  filter  it 
and  subject  it  to  a  polarization  test.  Through  the  action  of  the  benzoyl 
chloride  all  the  laevo-rotatory  substances  except  /3-oxybutyric  acid  will 
have  been  removed  and  the  ItEvo-rotation  now  exhibited  by  the  urine  will 
be  due  entirely  to  that  acid. 

XIX.  Acidity. 

Folin's  Method. — The  total  acidity  of  urine  may  be  determined  as 
follows:  Place  25  c.c.  of  urine  in  a  200  c.c.  Erlenmcyer  flask  and  add 

'  Shaffer  has  recently  called  attention  to  the  fact  that  it  is  extremely  difficult  to  extract 
all  of  the  crotonii  acid  if  but  three  extractions  are  made 

-  If  sugar  is  present  it  must  he  removed  by  fermentation. 

'  The  Schleicher  and  Schiill  fat-free  extraction  thimble  is  very  satisfactory. 


428  PHYSIOLOGICAL    CHEMISTRY. 

15-20  grams  of  finely  pulverized  potassium  oxalate  and  1-2  drops  of  a  i 
per  cent  phenolphthalein  solution  to  the  fluid.  Shake  the  mixture 
vigorously  for  1-2  minutes  and  titrate  it  immediately  with  N/io  sodium 
hydroxide  until  a  faint  but  unmistakable  pink  remains  permanent  on 
further  shaking.  Take  the  burette  reading  and  calculate  the  acidity  of 
the  urine  under  examination. 

Calculation. — If  y  represents  the  number  of  cubic  centimeters  of 
N/io  sodium  hydroxide  used  and  y'  represents  the  volume  of  urine 
excreted  in  twenty-four  hours,  the  total  acidity  of  the  twenty-four-hour 
urine  specimen  may. be  calculated  by  means  of  the  following  proportion: 

2~,:y::y' -.x  (acidity  of  24-hour  urine  expressed  in  cubic  centimeters  of 
N/io  sodium  hydroxide). 

Each  cubic  centimeter  of  N/io  sodium  hydroxide  contains  0.004 
gram  of  sodium  hydroxide,  and  this  is  equivalent  to  0.0063  gram  of 
oxalic  acid.  Therefore,  in  order  to  express  the  total  acidity  of  the 
twenty-four-hour  urine  specimen  in  equivalent  grams  of  sodium  hydroxide, 
multiply  the  value  of  x,  as  just  determined,  by  0.004,  or  multiply  the 
value  of  X  by  0.0063  i^  i^  i^  desired  to  express  the  total  acidity  in  grams 
of  oxalic  acid. 

XX.  Purine  Bases. 

I.  Welker's  Modification  of  the  Methods  of  Arnstein  and  of 
Salkowski.' — -Four  hundred  cubic  centimeters  of  urine,  free  from 
protein,  are  treated  with  100  c.c.  of  magnesia  mixture  and  600  c.c.  of 
water.  This  is  then  filtered  and  of  the  clear  filtrate  a  measured  quantity 
(600-800  c.c.)  is  treated  with  an  excess  (10  c.c.)  of  a  3  per  cent  silver 
nitrate  solution.  Concentrated  ammonium  hydroxide  is  added  in 
small  quantities,  with  stirring,  until  all  the  chlorides  have  dissolved. 
Allow  the  flocculent  precipitate  of  the  silver  purine  compounds  to  settle 
to  the  bottom,  then  pass  the  supernatant  liquid  through  the  filter  before 
disturbing  the  precipitate.  Finally  transfer  the  precipitate  quantita- 
tively to  the  paper  which  must  be  of  known  nitrogen  content.  The 
precipitate  is  washed  with  dilute  (r  per  cent)  ammonium  hydroxide. 
The  paper  with  the  precipitate  is  then  transferred  to  a  Kjeldahl  flask 
and  about  100  c.c.  of  water  and  a  small  quantity  (about  o.i  gram)  of 
magnesium  oxide  are  added.  The  water  is  then  boiled  until  all  the 
ammonia  has  been  driven  off.     Test  the  steam  with  litmus  paper. 

The  material  in  the  flask  is  then  digested  by  means  of  the  usual 
Kjeldahl  method  (see  p.  401).     The  digestion  must  be  watched  care- 

'  Private  communication  from  Dr.  W.  H.  Welker. 


urine:  quantitative  analysis.  429 

fully  at  the  time  the  sulphuric  acid  reaches  sufficient  concentration  to 
affect  the  filter  paper,  inasmuch  as  the  SOj  produced  causes  consider- 
able frothing.  The  total  nitrogen  (purine  base,  uric  acid  and  filter- 
paper  nitrogen)  is  now  determined  in  the  usual  way  (see  Kjeldahl  Method, 
p.  401).  This  result  minus  the  uric  acid  and  filter-paper  nitrogen  will 
give  the  figure  for  the  purine-base  nitrogen. 

2.  Kriiger.  and  Schmidt's  Method. — This  method  serves  for  the 
determination  of  both  uric  acid  and  the  purine  bases.  The  principle 
involved  is  the  precipitation  of  both  the  uric  acid  and  the  purine  bases 
in  combination  with  copper  oxide  and  the  subsequent  decomposition 
of  this  precipitate  by  means  of  sodium  sulphide.  The  uric  acid  is  then 
precipitated  by  means  of  hydrochloric  acid  and  the  purine  bases  are 
separated  from  the  filtrate  in  the  form  of  their  copper  or  silver  com- 
pounds. The  nitrogen  content  of  the  precipitates  of  uric  acid  and 
purine  bases  is  then  determined  by  means  of  the  Kjeldahl  method  (see 
p.  401)  and  the  corresponding  values  for  uric  acid  and  purine  bases 
calculated.  The  method  is  as  follows:  To  400  c.c.  of  albumin-free 
urine  ^  in  a  liter  flask,'  add  24  grams  of  sodium  acetate,  40  c.c.  of  a  solu- 
tion of  sodium  bisulphite'  and  heat  the  mixture  to  boiling.  Add  40- 
80  c.c.^  of  a  10  per  cent  solution  of  copper  sulphate  and  maintain  the 
temperature  of  the  mixture  at  the  boiling-point  for  at  least  three  minutes. 
Filter  off  the  flocculent  precipitate,  wash  it  with  hot  w^ater  until  the 
wash  water  is  colorless,  and  return  the  washed  precipitate  to  the  flask 
by  puncturing  the  tip  of  the  filter  paper  and  washing  the  precipitate 
through  by  means  of  hot  water.  Add  water  until  the  volume  in  the 
flask  is  approximately  200  c.c,  heat  the  mixture  to  boiling  and  decom- 
pose the  precipitate  of  copper  oxide  by  the  addition  of  30  c.c.  of  sodium 
sulphide  solution.^  After  decomposition  is  complete,  the  mixture 
should  be  acidified  with  acetic  acid  and  heated  to  boiling  until  the  sepa- 
rating sulphur  collects  in  a  mass.  Filter  the  hot  fluid  by  means  of  a  filter- 
pump,  wash  with  hot  water,  add  10  c.c.  of  10  per  cent  hydrochloric  acid 
and  evaporate  the  filtrate  in  a  porcelain  dish  until  the  total  volume  has 
been  reduced  to  about  10  c.c.  Permit  this  residue  to  stand  about  two 
hours  to  allow'  for  the  separation  of  the  uric  acid,  leaving  the  purine 

'  If  albumin  is  present,  the  urine  should  be  heated  to  boiling,  acidified  with  acetic  acid, 
and  filtered. 

-  The  total  volume  of  urine  for  the  twenty-four  hours  should  be  sufficiently  diluted  with 
water  to  make  the  total  volume  of  the  solution  1600-2000  c.c. 

'  A  solution  containing  50  grams  of  Kahlbaum's  commercial  sodium  bisulphite  in  100  c.c. 
of  water. 

*  The  exact  amount  depending  upon  the  content  of  the  purine  bases. 

*  This  is  made  by  saturating  a  i  per  cent  solution  of  sodium  hydroxide  with  hydrogen 
sulphide  gas  and  adding  an  equal  volume  of  i  per  cent  sodium  hydroxide. 

Ordinarily  the  addition  of  30  c.c.  of  this  solution  is  sufficient,  but  the  presence  of  an  excess 
of  sulphide  should  be  proven  by  adding  a  drop  of  lead  acetate  to  a  drop  of  the  solution.  Under 
these  conditions  a  dark  brown  color  will  show  the  presence  of  an  excess  of  sodium  sulphide. 


43©  PHYSIOLOGICAL   CHEMISTRY. 

bases  in  solution.  Filter  off  the  precipitate  of  uric  acid,  using  a  small 
filter  paper,  and  wash  the  uric  acid,  with  water  made  acid  with  sulphuric 
acid,  until  the  total  volume  of  the  original  filtrate  and  the  wash  water 
aggregates  75  c.c.  Determine  the  nitrogen  content  of  the  precipitate  by 
means  of  the  Kjeldahl  method  (see  p.  401),  and  calculate  the  uric  acid 
equivalent.  ^ 

Render  the  filtrate  from  the  uric  acid  crystals  alkaline  with  sodium 
hydroxide,  add  acetic  acid  until  faintly  acid  and  heat  to  70°  C.  Now 
add  I  c.c.  of  a  10  per  cent  solution  of  acetic  acid  and  10  c.c.  of  a  sus- 
pension of  manganese  dioxide^  to  oxidize  the  traces  of  uric  acid  which 
remain  in  the  solution.  Agitate  the  mixture  for  one  minute,  add  10  c.c. 
of  the  sodium  bisulphite  solution^  and  5  c.c.  of  a  10  per  cent  solution  of 
copper  sulphate  and  heat  the  mixture  to  boiling  for  three  minutes.  Filter 
off  the  precipitate,  wash  it  with  hot  water,  and  determine  its  nitrogen 
content  by  means  of  the  Kjeldahl  method  (see  p.  401).  Inasmuch  as  the 
composition  and  proportion  of  the  purine  bases  present  in  urine  is  variable, 
no  factor  can  be  applied.  The  result  as  regards  these  bases  must  there- 
fore be  expressed  in  terms  of  nitrogen. 

Benedict  and  Saiki*  report  cases  in  which  the  total  purine  nitrogen 
by  this  method  was  less  than  the  uric-acid  nitrogen  as  determined  by 
the  Folin-Shaffer  method.  The  inaccuracy  was  found  to  lie  in  the 
Kriiger  and  Schmidt  method.  To  obviate  this  they  advise  the  addition 
of  20  c.c.  of  glacial  acetic  acid  for  each  300  c.c.  of  urine  employed,  the 
acid  being  added  before  the  first  precipitation. 

3.  Salkowski's  Method. — ^Place  400-600  c.c.  of  protein-free  urine 
in  a  beaker.  Introduce  into  another  beaker  30-50  c.c.  of  an  ammoni- 
acal  silver  solution^  with  30-50  c.c.  of  magnesia  mixture, °  add  some 
ammonium  hydroxide  and  if  necessary  some  ammonium  chloride  to 
clear  the  solution.  Now  add  this  solution  to  the  urine,  stirring  con- 
tinually with  a  glass  rod,  and  allow  the  mixture  to  stand  for  one-half 
hour.  Collect  the  precipitate  on  a  filter  paper,  wash  it  with  dilute 
ammonium  hydroxide,  and  finally  wash  it  back  into  the  original  beaker. 
Suspend  the  precipitate  in  600-800  c.c.  of  water,  add  a  few  drops  of 
hydrochloric  acid  and  decompose  it  by  means  of  hydrogen  sulphide. 

'  This  may  be  done  by  multiplying  the  nitrogen  value  by  three  and  adding  three  and 
one-half  milligrams  to  the  product  as  a  correction  for  the  uric  acid  remaining  in  solution 
in  the  75  c  c. 

^  Made  by  heating  a  0.5  per  cent  solution  of  potassium  permanganate  with  a  little  alcohol 
until  it  is  decolorized. 

'  To  dissolve  the  excess  of  manganese  dioxide. 

*  Benedict  and  Saiki:    Jour.  Biol.  Chetn.,  7,  27,  1909. 

'•"  Prepared  by  dissolving  26  grams  of  silver  nitrate  in  about  500  c.c.  of  water,  adding  enough 
ammonium  hydroxide  to  redissolve  the  prec  ipitate  which  forms  upon  the  first  addition  of  the 
ammonia  and  making  the  balance  of  the  mixture  up  to  i  liter  with  water. 

•  Directions  for  preparation  may  be  found  on  page  313. 


urine:  quantitative  analysis. 


431 


Now  heat  the  solution  to  boiling,  filter  while  hot  and  evaporate  the 
filtrate  to  dryness  on  a  water-bath.  Extract  the  residue  with  20-30  c.c. 
of  hot  3  per  cent  sulphuric  acid  and  allow  the  extract  to  stand  twenty- 
four  hours.  Filter  off  the  uric  acid,  wash  it,  make  the  filtrate  ammoni- 
acal  and  precipitate  the  purine  bases  again  with  silver  nitrate.  Collect 
this  precipitate  on  a  small-sized  chlorine-free  filter  paper,  wash,  dry, 
and  incinerate  it  in  the  usual  manner.  Now  dissolve  the  ash  in  nitric 
acid  and  titrate  with  ammonium  thiocyanate  according  to  the  Volhard- 
Arnold  method  (see  p.  419).  Calculate  the  content  of  purine  bases  in 
the  urine  examined,  bearing  in  mind  that  in  an  equal  mixture  of  the 
silver  salts  of  the  purine  bases,  such  as  we  have  here,  one  part  of  silver 
corresponds  to  0.277  gram  of  nitrogen  or  to  0.7381  gram  of  the  bases. 

XXI.  Purine  Nitrogen. 


Hall's  Purinometer.'^ — By  means  of  the  instrument  shown  in  Fig. 
133,  urine  may  be  examined  for  total  purine  nitrogen,  i.  e.,  nitrogen  in 
the  form  of  purine  bases,  urates  and  uric  acid.  The  method  does 
not  give  an  absolutely  accurate  measure  of  the  purine 
values.  It  is,  however,  of  considerable  service  clini- 
cally. The  principle  of  the  method  is  the  preliminary 
precipitation  of  the  phosphates  present  followed  by  the 
precipitation  of  the  purine  bodies  in  the  form  of  their 
silver  compounds  by  means  of  an  ammoniacal  silver 
nitrate  solution.  The  volume  of  this  silver  precipitate 
is  then  determined  and  its  nitrogen  value  interpolated 
by  means  of  a  table  of  equivalent  values.  In  using 
the  purinometer  proceed  as  follows:  Collect  the 
twenty-four-hour  urine  and  mix  it  thoroughly.  Take 
TOO  c.c.  of  the  urine  and  if  albumin  is  present  make 
slightly  acid  with  acetic  acid  and  boil  and  filter. 
Close  the  stopcock  of  the  instrument  and  introduce 
Qo  c.c.  of  urine  and  20  c.c.  of  a  modified  magnesia 
mixture.*  Turn  the  stopcock  and  permit  the  pre- 
cipitated phosphates  to  pass  into  the  lower  chamber 
of  the  instrument.  After  an  interval  of  ten  minutes 
has  elapsed  the  stopcock  should  be  closed  and  suffi- 


FiG.  133. — Hall's 
Purinometer. 


'  Hall:  The  Purine  Bodies,  Philadelphia,  1904. 

-This  is  prepared  as  follows:  Dissolve  10  grams  of  magnesium  chloride  in  about  75  c.c. 
of  water  and  add  10  grams  of  ammonium  chloride.  Introduce  100  c.c.  of  concentrated 
ammonium  hydroxide  into  this  mixture  If  a  precipitate  forms  add  ammonium  hydroxide 
until  a  clear  solution  is  obtained.  Make  the  volume  200  c.c.  by  means  of  water  and  add  10 
grams  of  purified  talcum. 


432  PHYSIOLOGICAL    CHEMISTRY. 

cicnt  ammoniacal  silver  nitrate  solution^  added  to  make  the  total 
volume  in  the  upper  chamber  loo  c.c.  The  precipitate  of  the  silver 
compounds  of  the  purine  bodies  should  be  pale  yellow.  Any  silver 
chloride  present  may  be  brought  into  solution  in  the  strong  ammoniacal 
solution  by  the  repeated  inversion  of  the  purinometer.  In  case  the  chloride 
does  not  dissolve  it  should  be  brought  into  solution  by  the  addition  of 
further  ammonium  hydroxide.  Place  the  purinometer  in  a  dark  room 
for  twenty-four  hours  and  at  the  end  of  this  time  read  the  volume  of  the 
purine  precipitate.  Interpolate  the  value  in  terms  of  purine  nitrogen  by 
means  of  the  following  table: 

^      .   .  Purine  nitrogen 

Precipitate  p^^.  ^^^^ 

^''''  (grams  in  loo  c.c.) 

4 o . 0078 

2 o . 0097 

6 0.0117 

7 0.0136 

8 0.0156 

9 0.0175 

10 0.0185 

II 0.0195 

12 o . 0205 


13- 


0.0218 


14 0.0225 

15 0.0234 

16 0.0249 

xy 0.0260 

18 0.0265 

19 0.0270 

20 0.0275 

21 0.0283 

22 o . 0286 

23 0.0299 

24 0.0312 

25 0.0325 

Calculation. — Multiply  the  purine  nitrogen  percentage  by  the  total 
volume  of  urine  and  divide  by  100  to  obtain  the  total  purine  nitrogen 
value.  For  example,  if  the  precipitate  was  found  to  be  12  c.c.  and  the 
total  volume  of  the  twenty-four-hour  urine  was  1300  c.c.  the  calculation 
would  be  as  follows: 

12  c.c.  =0.0205  per  cent  purine  nitrogen. 

0.0205  X13.0  =0.2665  gram  purine  nitrogen. 


XXII.  Allantoin.^' 

Paduschka-Underhill-Kleiner  Method. — To  50-100  c.c.  of  urine 
m  a  beaker  add  basic  lead  acetate  until  no  more  precipitate  forms. 

'  This  solution  has  the  following  formula: 

Silver  nitrate i  gram 

Ammonium  hydroxide  (sp.  gr.  0.90) 100  c.c. 

Talcum 5  grams 

Distilled  water 100  c.c. 

'  A  much  more  accurate  method  has  been  devised  by  Wiechowski  {Biochemische  Zeitschrift, 
19,  368,  1909.) 


urine:  quantitative  analysis.  433 

Filter  and  pass  hydrogen  sulphide  gas  through  an  ahquot  portion  of  the 
filtrate  to  remove  the  excess  of  lead.  ^  Filter  again,  drive  off  the  hydrogen 
sulphide  by  heat  and  treat  an  aliquot  portion  of  the  filtrate  with  a  lo  per 
cent  solution  of  silver  nitrate  until  precipitation  is  complete."  Filter  oflf 
this  precipitate,  wash  it  with  water  and  determine  its  nitrogen  content  by 
means  of  the  Kjeldahl  method  (see  p.  401).  This  is  the  "purine  nitro- 
gen." Rendjer  an  aliquot  portion  of  the  filtrate  faintly  alkaline,^  with 
a  I  per  cent  solution  of  ammonium  hydroxide  and  add  50-100  c.c.  of  a  10 
per  cent  solution  of  silver  nitrate.  If  allantoin  be  present  a  white, 
flocculent  precipitate  will  form  and  gradually  sink  to  the  bottom  of  the 
solution.  Filter,  wash  the  precipitate  free  from  ammonium  hydroxide 
by  means  of  a  i  per  cent  solution  of  sodium  sulphate  and  determine  its 
nitrogen  content  by  the  Kjeldahl  method  (see  p.  401). 


XXIII.  Oxalic  Acid. 

Salkowski-Autenrieth  and  Barth  Method. — Place  the  twenty- 
four-hour  urine  specimen  in  a  precipitating  jar,  add  an  excess  of  calcium 
chloride,  render  the  urine  strongly  ammoniacal,  stir  it  well,  and  allow 
it  to  stand  18-20  hours.  Filter  off  the  precipitate,  wash  it  with  a  small 
amount  of  water  and  dissolve  it  in  about  30  c.c.  of  a  Jiot  15  per  cent 
solution  of  hydrochloric  acid.  By  means  of  a  separatory  funnel  extract 
the  solution  with  150  c.c.  of  ether  which  contains  3  per  cent  of  alcohol, 
repeating  the  extraction  four  or  five  times  with  fresh  portions  of  ether. 
Unite  the  ethereal  extracts,  allow  them  to  stand  for  an  hour  in  a  flask, 
and  then  filter  through  a  dry  filter  paper.  Add  5  c.c.  of  water  to  the  fil- 
trate, to  prevent  the  formation  of  diethyl  oxalate  when  the  solution  is 
heated,  and  distil  off  the  ether.  If  necessary,  decolorize  the  liquid  with 
animal  charcoal  and  filter.  Concentrate  the  filtrate  to  3-5  c.c,  add  a 
little  calcium  chloride  solution,  make  it  ammoniacal,  and  after  a  few 
minutes  render  it  slightly  acid  with  acetic  acid.  Allow  the  acidified 
solution  to  stand  several  hours,  collect  the  precipitate  of  calcium  oxalate 
on  a  washed  filter  paper,*  wash,  incinerate  strongly  (to  CaO),  and  weigh 
in  the  usual  manner. 

Calculation. — Since  56  parts  of  CaO  are  equivalent  to  90  parts  of 
oxalic  acid,  the  quantity  of  oxalic  acid  in  the  volume  of  urine  taken 
may  be  determined  by  multiplying  the  weight  of  CaO  by  the  factor 
1.607 1. 

'  In  the  original  method  of  Paduschka  sodium  sulphate  is  used  for  this  purpose. 
-  Ordinarily  from  20-30  c.c.  is  required 
'  Using  litmus  as  the  indicator. 
*  Schleicher  and  Schiill,  No.  589,  is  satisfactorj-. 
28 


434  PHYSIOLOGICAL    CHEMISTRY. 

XXIV.  Total  Solids. 

1.  Drying  Method. — Place  5  c.c.  of  urine  in  a  weighed  shallow 
dish,  acidify  very  slightly  with  acetic  acid  (1-3  drops),  and  dry  it  in 
vacuo  in  the  presence  of  sulphuric  acid  to  constant  weight.  Calculate 
the  percentage  of  solids  in  the  urine  sample  and  the  total  solids  for  the 
twenty-four-hour  period. 

Practically  all  the  methods  the  technique  of  which  includes  evapo- 
ration at  an  increased  temperature,  either  under  atmospheric  conditions 
or  in  vacuo,  are  attended  with  error. 

■  Shackell's  method^  which  entails  the  vacuum  desiccation  of  the  frozen 
sample  is  extremely  satisfactory  and  should  be  used  in  all  biological  work 
where  the  greatest  accuracy  is  desired. 

2.  Calculation  by  Long's  Coefficient. — The  quantity  of  solid 
material  contained  in  the  urine  excreted  for  any  twenty-four-hour  period 
may  be  approximately  computed  by  multiplying  the  second  and  third 
decimal  figures  of  the  specific  gravity  by  2.6.  This  gives  us  the  number 
of  grams  of  solid  matter  in  i  liter  of  urine.  From  this  value  the  total 
solids  for  the  twenty-four-hour  period  may  easily  be  determined. 

Calculation. — If  the  volume  of  urine  for  the  twenty-four  hours  was 
1120  c.c.  and  the  specific  gravity  1.018,  the  calculation  would  be  as 
follows: 

{a)      16  X2.6  =46.8  grams  of  solid  matter  in  i  liter  of  urine. 

46.8X1120 
(b)  =^2.4.  grams  of  solid  matter  in  11 20  c.c.  of  urine, 

icoo  ^       ^ 

Long's  coefficient  was  determined  for  urine  whose  specific  gravity 
was  taken  at  25°  C.  and  is  probably  more  accurate,  for  conditions 
obtaining  in  America,  than  the  older  coefficient  of  Haeser,  2.33. 

'  Shackell:  American  Journal  of  Physiology,  24,  325,  1909. 


CHAPTER  XXIII. 

QUANTITATIVE  ANALYSIS  OF  MILK,   GASTRIC  JUICE,   AND 

BLOOD. 


)C.C. 


(a)  Quantitative  Analysis  of  Milk. 

1.  Specific  Gravity. — This  may  be  determined  conveniently  by  means 
of  a  Soxhlet,  \'eith,  or  Quevenne  lactometer.  A  lactometer  reading  of 
32°  denotes  a  specific  gravity  of  1.032.  The  determination  should  be 
made  at  about  60°  F.  and  the  lactometer  reading  cor- 
rected by  adding  or  subtracting  0.1°  for  every  degree  F. 
above  or  below  that  temperature. 

Fat.  (a)  Babcock's  Centrifugal  Method. — The  princi- 
ple of  this  method  is  the  destruction  of  organic  matter 
other  than  fat  by  sulphuric  acid  and  the  centrifugation 
of  the  acid  solution  in  the  special  tube  shown  in  Fig. 
134  and  the  subsequent  reading  of  the  percentage  of  fat 
by  means  of  the  tube's  graduated  neck.  The  method  is 
one  of  the  most  satisfactory  in  common  use  and  is 
accurate  to  within  0.5  per  cent.  Proceed  as  follows: 
By  means  of  a  special  narrow  pipette  introduce  milk 
into  the  tube  up  to  the  5  c.c.  mark.  Now  add  sufficient 
sulphuric  acid  (sp.  gr.  i. 83-1. 834)  to  fill  the  body  of  the 
tube  and  rotate  the  tube  to  secure  a  homogeneous  acid- 
milk  solution.  Fill  the  neck  of  the  tube  with  an  acid- 
alcohol  mixture.^  Centrifuge  the  tube  and  contents  for 
one  to  two  minutes  and  read  off  the  percentage  of  fat  by 
means  of  the  graduated  neck  of  the  tube.  If  the  top 
of  the  fat  column  is  not  at  zero  it  may  be  brought  there 
by  the  addition  of  water  and  a  moment's  recentrifugation. 

In  case  very  rich  milk  (over  5  per  cent  fat)  is  under 
examination,  it  may  be  diluted  with  an  equal  volume 
of  water  before  examination  and  the  fat  percentage 
multiplied  by  2.  In  the  examination  of  cream  it  is 
customary  to  dilute  the  sample  with  four  volumes  of  water  and  multiply 
the  resultant  fat  value  by  5. 

2.  Fat. — (6)  Quantitative  Determination  of  Fat  in  Milk  by  the  Meigs^ 

'  This  mixture  consists  of  equal  volumes  of  amyl  alcohol  and  concentrated  hydrochloric 
acid. 

*  Original  paper  by  Dr.  Arthur  V.  Meigs  in  Philadelphia  Medical  Times,  July  i,  18S2. 

435 


Fig.  134. — Bab- 
cock  Tube. 


436 


PHYSIOLOGICAL   CHEMISTRY. 


Method  with  Modification  and  Improved  Apparatus  by  Croll.  ^ — The  method 
as  stated  by  Dr.  Meigs  is:  Approximately  lo  c.c.  of  milk  is  carefully 
weighed  and  transferred  to  an  ordinary  loo  c.c.  glass-stoppered  graduated 
cylinder.  Twenty  c.c.  each  of  distilled  water  and  ether  (0.720)  are 
added,  the  ground-glass  stopper  tightly  inserted  in  the  bottle,  and  the 

whole  shaken  vigorously  for  five  minutes. 
Then  the  bottle  is  carefully  unstoppered, 
20  c.c.  95  per  cent  alcohol  added,  the 
stopper  reinserted  and  again  shaken  for 
five  minutes.  The  bottle  is  now  placed 
on  a  table  and  the  contents  will  separate 
into  two  distinct  strata,  the  upper  of 
which  contains  practically  all  the  fat. 
This  stratum  is  carefully  removed  by  a 
small  pipette  and  transferred  to  a  carefully 
weighed  glass  evaporating  dish.  The  thin 
ether  layer  remaining  is  washed  by  the 
addition  of  5  c.c.  of  ether.  This  is  re- 
moved by  pipetting  off.  This  washing  is 
repeated  four  times.  On  each  addition 
the  sides  of  the  bottle  should  carefully  be 
washed  down  by  the  fresh  ether.  Finally, 
the  pipette  is  rinsed  with  a  little  ether. 
The  evaporating  dish  with  contents  is  now 
placed  on  a  safety  water-bath  and  the  ether 
evaporated.  The  drying  is  continued  in  a 
hot-air  oven  at  a  temperature  below  100° 
C.  and  finally  completed  in  a  desiccator  to 
constant  weight. 

Croll's  modification  consists  of  subse- 
quent repeated  extraction  of  the  end- 
product  of  evaporation  with  absolute  ether.  The  combined  extracts 
are  filtered  and  the  small  filter  paper  is  washed  repeatedly  with  absolute 
ether.  The  combined  extracts  and  washings  are  evaporated  and  dried 
as  before  and  then  weighed. 

The  piece  of  apparatus  shown  in  Fig.  135,  above  was  also  devised 
by  Croll  to  do  away  with  the  use  of  the  pipette.  On  closing  the  top 
with  a  finger  and  blowing  into  the  mouth-piece,  the  upper  stratum  is 
forced  out  into  the  dish.  The  bottle  is  washed  by  simply  pouring  the 
ether  into  the  tube.     This  lessens  the  possibility  of  accidental  loss. 

The  accuracy  of  the  method  compared  with  that  of  the  Soxhlet  method, 

*  Private  Communication. 


Fig.  135. — Croll's  Fat  Apparatus. 


QUANTITATIVE   ANALYSIS    Ok'   MILK. 


437 


using  the  paper-coil  modification  and  extracting  until  fresh  portions  of 
absolute  ether  gave  no  further  trace  of  extractive  material,  is  shown  by 
the  average  difference  on  twelve  samples  of  human  milk  being  only  0.017 
per  cent  less  than  by  the  Soxhiet  and  on  seven  samples  cow's  milk  being 
onlv  0.0  TQ  per  cent  less.  The  extreme  differences  in  case  of  the  human 
milk  were  —0.004  per  cent  and  —0.044  per 
cent  and  in  case  of  the  cow's  milk— 0.006 
per  cent  and— 0.068  per  cent. 

(c)  Adams'  Paper-coil  Method.- — Intro- 
duce about  5  c.c.  of  milk  into  a  small 
beaker,  quickly  ascertain  the  weight  to 
centigrams,  stand  a  fat-free  coil'  in  the 
beaker,  and  incline  the  vessel  and  rotate 
the  coil  in  order  to  hasten  the  absorption 
of  the  milk.  Immediately  upon  the  com- 
plete absorption  of  the  milk  remove  the 
coil  and  again  quickly  ascertain  the 
weight  of  the  beaker.  The  difference  in 
the  weights  of  the  beaker  at  the  two 
weighings  represents  the  quantity  of  milk 
absorbed  by  the  coil.  Dry  the  coil  care- 
fully at  a  temperature  below  100°  C.  and 
extract  it  with  ether  for  3-5  hours  in  a 
Soxhiet  apparatus  (Fig.  136,  p.  437.) 
Using  a  safety  water-bath,  heat  the  flask 
containing  the  fat  to  constant  weight  at  a 
temperature  below  100°  C. 

Calculatio-n. — Divide  the  weight  of  fat, 
in  grams,  by  the  weight  of  milk,  in 
grams.  The  quotient  is  the  percentage 
of  fat  contained  in  the  milk  examined. 

{d)  Approximate  Determination  by  Feser'sLactoscope. — Milk  is  opaque 
mainly  because  of  the  suspended  fat  globules  and  therefore  by  means 
of  the  estimation  of  this  opacity  we  may  obtain  data  as  to  the  approximate 
content  of  fat.  Feser's  lactoscope  (Fig.  137)  may  be  used  for  this  purpose. 
Proceed  as  follows:  By  means  of  the  graduated  pipette  accompanying 
the  instrument  introduce  4  c.c.  of  milk  into  the  lactoscope.  Add  water 
gradually,  shaking  after  each  addition,  and  note  the  point  at  which  the 
black  lines  upon  the  inner  white  glass  cylinder  are  distinctly  visible. 
Observe  the  point  on  the  graduated  scale  of  the  lactoscope  which  is  level 
with  the  surface  of  the  diluted  milk.     This  reading  represents  the  per- 

*  Verj-  satisfactorj'  coils  are  manufactured  by  Schleicher  and  Schull. 


SoxHLET  Apparatus. 


43S 


PHYSIOLOGICAL    CHEMISTRY. 


-I 


centage  of  fat  present  in  the  undiluted  milk.  Pure  milk  should  contain  at 
least  3  per  cent  of  fat. 

3.  Total  Solids/ — Introduce  2-5  grams  of  milk  into  a  weighed  fiat- 
bottomed  platinum  dish^  and  quickly  ascertain  the  weight  to  milligrams. 
Expel  the  major  portion  of  the  water  by  heating  the  open  dish  on  a  water- 
bath  and  continue  the  heating  in  an  air-bath  or  water  oven  at  97°-ioo°  C. 
until  the  weight  is  constant.  (If  platinum  dishes  are  employed  this  residue 
may  be  used  in  the  determination  of  ash  according  to  the  method  described 
below.) 

Calculation} — Divide  the  weight  of  the  residue,  in  grams,  by  the 
weight  of  milk  used,  in  grams.  The  quotient  is  the 
percentage  of  solids  contained  in  the  milk  examined. 

4.  Ash. — Heat  the  dry  solids  from  2-5  grams  of 
milk,  obtained  according  to  the  method  just  given,  over 
a  very  low  flame*  until  a  white  or  light  gray  ash  is  ob- 
tained. Cool  the  dish  in  a  desiccator  and  weigh.  (This 
ash  may  be  used  in  testing  for  preservatives  according  to 
directions  on  page  244.) 

5.  Proteins. — Introduce  a  known  weight  of  milk 
(5-10  grams)  into  a  500  c.c.  Kjeldahl  digestion  flask 
and  add  20  c.c.  of  concentrated  sulphuric  acid  and 
about  0.2  gram  of  copper  sulphate.  Expel  the  major 
portion  of  the  water  by  heating  over  a  low  flame  and 
finally  use  a  full  flame  and  allow  the  mixture  to  boil  1-2 

hours.  Complete  the  determination  according  to  the  directions  given 
under  Kjeldahl  Method,  page  401. 

Calculation. — Multiply  the  total  nitrogen  content  by  the  factor  6.37^ 
to  obtain  the  protein  content  of  the  milk  examined. 

6.  Caseinogen. — Mix  about  20  grams  of  milk  with  40  c.c.  of  a 
saturated  solution  of  magnesium  sulphate  and  add  the  salt  in  substance 
until   no   more   will   dissolve.     The  precipitate  consists  of  caseinogen 

'  Shackell's  method  for  the  vacuum  desiccation  of  frozen  preparations  may  be  used  where 
great  accuracy  is  desired  (see  American  Journal  of  Physiology,  24,  325,  1909). 

2  Lead  foil  dishes,  costing  only  about  one  dollar  per  gross,  make  a  very  satisfactory  substi- 
tute for  the  platinum  dishes. 

^  The  percentage  of  total  solids  may  be  calculated  from  the  specific  gravity  and  percentage 
of  fat  by  means  of  the  following  formula  which  has  been  proposed  by  Richmond: 

5=0.25  L  +  1.2  F4-O.I4 
S  =  total  solids. 
L  =  lacometer  reading. 
F  =  fat  content. 

*  Great  care  should  be  used  in  this  ignition,  the  dish  at  no  time  being  heated  above  a  faint 
redness,  as  chlorides  may  volatilize. 

■''  The  usual  factor  employed  for  the  calculation  of  protein  from  the  nitrogen  content  is 
6.25  and  is  based  on  the  assumption  that  proteins  contain  on  the  average  16  per  cent  of  nitrogen. 
This  special  factor  of  6.37  is  used  here  to  calculate  the  protein  content  from  the  total  nitrogen, 
since  the  principal  protein  constituents  of  milk,  i.  e.,  caseinogen  and  laclalbumin ,  contain  15.7 
per  cent,  of  nitrogen. 


J 

Fig.  137. — Feser's 
Lactoscope. 


QUANTITATIVK    ANALYSIS    OF    MILK.  439 

admixed  with  a  little  fat  and  lacto-globulin.  Filter  off  the  precipitate, 
wash  it  thoroughly  with  a  saturated  solution  of  magnesium  sulphate/ 
transfer  the  filter  paper  and  precipitate  to  a  Kjeldahl  digestion  flask,  and 
determine  the  nitrogen  content  according  to  the  directions  given  in  the 
previous  experiment. 

Calculation. — Multiply  the  total  nitrogen  by  the  factor  6.37  to  obtain 
the  casein  content. 

7.  Hart's  Caseinogen  Method.- — Introduce  10.5  c.c.  of  milk  into 
a  200  c.c.  Erlcnmcyer  llask  and  add  75  c.c.  of  distilled  water  and  1-1.5 
c.c.  of  10  per  cent  acetic  acid.^  Mix  the  contents  by  giving  the  flask  a 
vigorous  rotary  motion.  The  precipitated  caseinogen  is  now  filtered  off 
upon  a  9-1 1  cm.  filter  paper.^  Wash  out  the  adsorbed  and  loosely 
combined  acetic  acid  by  means  of  cold  water.  Continue  the  washing 
of  both  the  caseinogen  on  the  filter  and  that  adhering  to  the  flask,  until 
the  wash  water  has  reached  a  volume  of  at  least  250  c.c. 

Now  return  the  precipitate  and  paper  to  the  original  Erlenmeyer  flask, 
add  75-80  c.c.  of  neutral  (carbon  dioxide-free)  water,  10  c.c.  of  N/io 
potassium  hydroxide  and  a  few  drops  of  phenolphthalein.  Stopper  the 
flask  and  shake  it  vigorously,  by  hand  or  machine,  until  the  caseinogen 
has  been  brought  into  solution.*  Rinse  the  stopper  with  neutral  (carbon 
dio.xide-free)  water  and  titrate  the  alkaline  caseinogen  solution  at  once 
with  N/io  hydrochloric  acid  until  there  is  a  disappearance  of  all  red 
color.  ° 

Calculation. — Subtract  the  corrected^  acid  reading  from  the  10  c.c. 
of  alkali  used.  The  difference  is  the  percentage  of  caseinogen  in  the 
milk.  For  example,  if  it  takes  6.7  c.c.  of  N/io  hydrochloric  acid  to 
titrate  the  alkaline  solution  to  the  end  point  and  the  check  test  was 
equivalent  to  0.2  c.c.  N/  10  acid  the  caseinogen  value  would  be  obtained 
as  follows: 

10 —(6.7  +  0.2)  =3.1  per  cent  caseinogen. 

8.  Lactalbumin. — To  the  filtrate  and  washings  from  the  determi- 

'  Preserve  the  filtrate  and  washings  for  the  determination  of  lactalbumin  (Expt.  8). 

-  Hart:  Jour.  Biol.  Client.,  6,  445,  1909. 

'  In  general  1.5  c.c.  of  acetic  acid  gives  a  clear  solution  which  filters  nicely  but  occasionally, 
when  the  milk  has  a  low  caseinogen  value  it  is  advisable  to  use  less  acetic  acid. 

*  The  process  of  filtration  may  be  retarded  through  the  packing  of  the  caseinogen  mass  upon 
the  filter  paper.  In  this  case  conduct  a  fine  stream  of  cold  water  against  the  upper  point  of 
contact  of  filter  paper  and  caseinogen.  By  this  means  the  caseinogen  precipitate  is  loosened 
and  gathers  in  the  apex  of  the  filter.  This  procedure  is  very  essential.  It  is  not  necessary  to 
remove  the  caseinogen  which  adheres  to  the  interior  of  the  flask. 

^  Solution  is  indicated  by  the  disappearance  of  the  white  caseinogen  particles  which  would 
otherwise  settle  to  the  bottom  of  the  flask. 

'  .\  check  test  should  be  run  parallel  with  the  entire  determination.  Even  with  special 
precautions  as  to  neutrality,  it  is  generally  found  that  an  acid  check  of  0.2-0.3  c.c.  will  be 
obtained.     This  check  titration  should  be  added  to  the  volume  of  acid  used  in  titration. 


440  PHYSIOLOGICAL    CHEMISTRY. 

nation  of  caseinogen,  in  Experiment  6,  add  Almen's^  reagent  until  no 
more  precipitate  forms.  Filter  off  the  precipitate  and  determine  the 
nitrogen  content  according  to  the  directions  given  under  Proteins,  above. 

Calculation. — Multiply  the  total  nitrogen  by  the  factor  6.37  to  obtain 
the  lactalbumin  content. 

8.  Lactose. — To  about  350  c.c.  of  water  in  a  beaker  add  20  grams 
of  milk,  mix  thoroughly,  acidify  the  fluid  with  about  2  c.c.  of  10  per  cent 
acetic  acid  and  stir  the  acidified  mixture  continuously  until  a  flocculent 
precipitate  forms.  At  this  point  the  reaction  should  be  distinctly  acid 
to  litmus.  Heat  the  solution  to  boiling  for  one-half  hour,  filter,  rinse 
the  beaker  thoroughly,  and  wash  the  precipitated  proteins  and  the 
adherent  fat  with  hot  water.  Combine  the  filtrate  and  wash  water  and 
concentrate  the  mixture  to  about  150  c.c.  Cool  the  solution  and  dilute 
it  to  200  c.c.  in  a  volumetric  flask.  Titrate  this  sugar  solution  according 
to  directions  given  under  Fehling's  Method,  page  384.  I'  'I 

Calculation. — Make  the  calculation  according  to  directions  given  under 
Fehling's  Method,  p.  384,  bearing  in  mind  that  10  c.c.  of  Fehling's  solution 
is  completely  reduced  by  0.0676  grams  of  lactose. 

(b)  Quantitative  Analysis  of  Gastric  Juice. 

Topfer's  Method. 

This  method  is  much  less  elaborate  than  many  others  but  is  sufficiently 
accurate  for  ordinary  clinical  purposes.  The  method  embraces  the  volu- 
metric determination  of  (i)  total  acidity,  (2)  combined  acidity,^  and  (3) 
free  acidity,  and  the  subsequent  calculation  of  (4)  acidity  due  to  organic 
acids  and  acid  salts,  from  the  data  thus  obtained. 

Strain  the  gastric  contents  and  introduce  10  c.c.  of  the  strained 
material  into  each  of  three  small  beakers  or  porcelain  dishes.^  Label  the 
vessels  A,  B  and  C,  respectively,  and  proceed  with  the  analysis  according 
to  the  directions  given  below. 

I.  Total  Acidity.^ — Add  3  drops  of  a  i  per  cent  alcoholic  solution 
of  phenolphthalein^  to  the  contents  of  vessel  A  and  titrate  with  N/io 
sodium  hydroxide  solution  until  a  faint  pink  color  is  produced  which 
cannot  be  deepened  by  further  addition  of  a  drop  of  N/io  sodium 
hydroxide.     Take  the  burette  reading  and  calculate  the  total  acidity. 

Calculation. — The  total  acidity  may  be  expressed  in  the  following  ways : 

*  Alm6n's  reagent  may  be  prepared  by  dissolving  5  grams  of  tannin  in  240  c.c.  of  50  per 
cent  alcohol  and  adding  10  c.c.  of  25  per  cent  acetic  acid. 

^  For  a  discussion  of  combined  acid  see  chapter  on  Gastric  Digestion. 

^  If  suflScient  gastric  juice  is  not  available  it  may  be  diluted  with  water  or  a  smaller  amount, 
e.  g.,  5  c.c.  taken  for  each  determination. 

*  This  includes  free  and  combined  acid  and  acid  salts. 

'  One  gram  of  phenolphthalein  dissolved  in  100  c.c.  of  95  per  cent  alcohol. 


QUANTITATIVE  ANALYSIS    OF    MILK.  44 1 

1.  The  number  of  cubic  centimeters  of  N/io  sodium  hydroxide 
solution  necessary  to  neutralize  100  c.c.  of  gastric  juice. 

2.  The  weight  (in  grams)  of  sodium  hydroxide  necessary  to  neutralize 
TOO  c.c.  of  gastric  juice. 

3.  The  weight  (in  grams)  of  hydrochloric  acid  which  the  total  acidity 
of  100  c.c.  of  gastric  juice  represents,  /.  e.,  percentage  of  hydrochloric  acid. 

The  forms  of  expression  most  frequently  employed  are  i  and  3, 
preference  bemg  given  to  the  former. 

In  making  the  calculation  note  the  number  of  cubic  centimeters  of 
N/io  sodium  hydroxide  required  to  neutralize  10  c.c.  of  the  gastric 
juice  and  multiply  it  by  10  to  obtain  the  number  of  cubic  centimeters 
necessary  to  neutralize  100  c.c.  of  the  fluid.  If  it  is  desired  to  express 
the  acidity  of  100  c.c.  of  gastric  juice  in  terms  of  hydrochloric  acid,  by 
weight,  multiply  the  value  just  obtained  by  0.00365.^ 

2.  Combined  Acidity." — Add  3  drops  of  sodium  alizarin  sulphonate 
solution^  to  the  contents  of  vessel  5  and  titrate  with  N/io  sodium  hydroxide 
solution  until  a  violet  color  is  produced.  In  this  titration  the  red  color, 
which  appears  after  the  tinge  of  yellow  due  to  the  addition  of  the  indicator 
has  disappeared,  must  be  entirely  replaced  by  a  distinct  violet  color. 
Take  the  burette  reading  and  calculate  the  combined  acidity. 

Calculation. — Since  the  indicator  used  reacts  to  all  acidities  except 
combined  acidity,  in  order  to  determine  the  number  of  cubic  centimeters 
of  N/io  sodium  hydroxide  necessary  to  neutralize  the  combined  acidity 
of  10  c.c.  of  the  gastric  juice,  we  must  subtract  the  burette  reading  just 
obtained  from  the  burette  reading  obtained  in  the  determination  of  the 
total  acidity.  The  data  for  100  c.c.  of  gastric  juice  may  be  calculated 
according  to  the  directions  given  under  Total  Acidity,  page  440. 

3.  Free  Acidity/ — Add  4  drops  of  di-methyl-amino-azobenzene 
(Topfer's  reagent)  solution^  to  the  contents  of  the  vessel  C  and  titrate 
with  N/io  sodium  hydroxide  solution  until  the  initial  red  color  is  replaced 
by  lemon  yellow.^  Take  the  burette  reading  and  calculate  the  free 
acidity. 

Calculatimi. — The  indicator  used  reacts  only  to  free  acid,  hence 
the  number  of  cubic  centimeters  of  N/io  sodium  hydroxide  used  in- 
dicates the  volume  necessary  to  neutralize  the  free  acidity  of  10  c.c.  of 
gastric  juice.  To  determine  the  data  for  100  c.c.  of  gastric  juice  proceed 
according  to  the  directions  given  under  Total  Acidity,  page  440. 

'  One  c.c.  of  N/io  hydrochloric  acid  contains  0.00365  gram  of  hydrochloric  acid. 

-  Hydrochloric  acid  combined  with  protein  material. 

'  One  gram  of  sodium  alizarin  sulphonate  dissolved  in  100  c.c.  of  water. 

*  Hydrochloric  acid  not  combined  with  protein  material. 

^  One-half  gram  dissolved  in  100  c.c.  of  95  per  cent  alcohol. 

•  If  the  lemon  yellow  color  appears  as  soon  as  the  indicator  is  added  it  denotes  the  absence 
of  free  acid. 


442  PHYSIOLOGICAL   CHEMISTRY. 

4.  Acidity  Due  to  Organic  Acids  and  Acid  Salts. — This  value 
may  be  conveniently  calculated  by  subtracting  the  number  of  cubic 
centimeters  of  N/io  sodium  hydroxide  used  in  neutralizing  the  contents 
of  vessel  C  from  the  number  of  cubic  centimeters  of  N/io  sodium  hydrox- 
ide solution  used  in  neutralizing  the  contents  of  vessel  B.  The  remainder 
indicates  the  number  of  cubic  centimeters  of  N/io  sodium  hydroxide 
solution  necessary  to  neutralize  the  acidity  due  to  organic  acids  and 
acid  salts  present  in  10  c.c.  of  gastric  juice.  The  data  for  100  c.c.  of 
gastric  juice  may  be  calculated  according  to  directions  given  under 
Total  Acidity,  page  440. 

(c)  Quantitative  Analysis  of  Blood. 

For  the  methods  involved  in  the  quantitative  examination  of  blood 
see  Chapter  XII. 


APPENDIX. 

Almen's  Reagent/ — Dissolve  5  grams  of  tannin  in  240  c.c.  of  50  per 
cent  alcohol  and  add  10  c.c.  of  25  per  cent  acetic  acid. 

Ammoniacal  Silver  Solution.' — Dissolve  26  grams  of  silver  nitrate 
in  about  500  c.c.  of  water,  add  enough  ammonium  hydroxide  to  redis- 
solve  the  precipitate  which  forms  upon  the  first  addition  of  the  ammonium 
hydroxide  and  make  the  volume  of  the  mixture  up  to  i  liter  with  water. 

Arnold-Lipliawsky  Reagent.^ — This  reagent  consists  of  two  definite 
solutions  which  are  ordinarily  preserved  separately  and  mixed  just  before 
using.     The  two  solutions  are  prepared  as  follows: 

(a)  One  per  cent  aqueous  solution  of  potassium  nitrite. 

(b)  One  gram  of  /'-amino-acetophenon  dissolved  in  100  c.c.  of  distilled 
water  and  enough  hydrochloric  acid  (about  2  c.c.)  added  drop  by  drop,  to 
cause  the  solution,  which  is  at  first  yellow,  to  become  entirely  colorless. 
An  excess  of  acid  must  be  avoided. 

Barfoed's  Solution. '—Dissolve  4.5  grams  of  neutral,  crystallized 
copper  acetate  in  100  c.c.  of  water  and  add  1.2  c.c.  of  50  per  cent  acetic 
acid. 

Baryta  Mixture.' — A  mixture  consisting  of  one  volume  of  a  saturated 
solution  of  barium  nitrate  and  two  volumes  of  a  saturated  solution  of 
barium  hydroxide. 

Basic  Lead  Acetate  Solution." — This  solution  possesses  the  following 
formula: 

Lead  acetate 180  grams. 

Lead  oxide  (Litharge) no  grams. 

Distilled  water  to  make 1000  grams. 

Dissolve  the  lead  acetate  in  about  700  c.c.  of  distilled  water,  with  boiling. 
Add  this  hot  solution  to  the  finely  powdered  lead  oxide  and  boil  for  one- 
half  hour  with  occasional  stirring.  Cool,  filter  and  add  sufl&cient  dis- 
tilled water  to  the  filtrate  to  make  the  weight  one  kilogram. 

Benedict's    Solutions.^ — First    Modification. — Benedict's    modified 

'  Ott's  precipitation  test,  p.  339.     Determination  of  lactalbumin,  p.  439. 

*  Salkowski's  method,  page  430. 

'  Arnold-Lipliawsky  reaction,  page  349. 

*  Barfoed's  test,  pages  36  and  331. 

'  Isolation  of  urea  from  urine,  page  287. 

*  Indican  determination,  page  416. 

'  Benedict's  modifications  of  Fehling's  test,  pages  328  and  329,  and  Benedict's  Method  No. 
I,  page  385. 

443 


444  PHYSIOLOGICAL  CHEMISTRY. 

Fehling  solution  consists  of  two  definite  solutions  — a  copper  sulphate 
solution  and  an  alkaline  tartrate  solution,  which  may  be  prepared  as 
follows: 

Copper  sulphate  solution  =34.65  grams  of  copper  sulphate  dissolved 
in  water  and  made  up  to  500  c.c. 

Alkaline  tartrate  solution  =100  grams  of  anhydrous  sodium  carbonate 
and  173  grams  of  Rochelle  salt  dissolved  in  water  and  made  up  to  100 
c.c. 

These  solutions  should  be  preserved  separately  in  rubber-stoppered 
bottles  and  mixed  in  equal  volumes  when  needed  for  use.  This  is  done 
to  prevent  deterioration. 

Second  Modification.- — \'ery  recently  Benedict  has  further  modified 
his  solution  and  has  succeeded  in  obtaining  one  which  does  not  deteriorate 
upon  long  standing.     It  has  the  following  composition: 

Copper  sulphate 17.3  grams. 

Sodium  citrate 173°  grams. 

Sodium  carbonate loo  .o  grams. 

Distilled  water  to  make  i  liter. 

With  the  aid  of  heat  dissolve  the  sodium  citrate  and  carbonate  in  about 
600  c.c.  of  water.  Pour  (through  a  folded  filter  paper  if  necessary)  into 
a  glass  graduate  and  make  up  to  850  c.c.  Dissolve  the  copper  sulphate 
in  about  100  c.c.  of  water  and  make  up  to  150  c.c.  Pour  the  corbonate- 
citrate  solution  into  a  large  beaker  or  casserole  and  add  the  copper 
sulphate  solution  slowly,  with  constant  stirring.  The  mixed  solution  is 
ready  for  use  and  does  not  deteriorate  upon  long  standing. 

Benedict's  solution  as  used  in  the  quantitative  determination  of  sugar 
(Method  No.  i)  consists  of  three  separate  solutions,  the  two  mentioned 
under  First  Modification  and  in  addition  a  potassium  ferro-thiocyanate 
solution.  This  third  solution  contains  15  grams  of  potassium  ferrocyanide, 
62.5  grams  of  potassium  thiocyanate  and  50  grams  of  anhydrous  sodium 
carbonate  dissolved  in  water  and  made  up  to  500  c.c.  In  preparing  the 
Benedict's  solution  for  quantitative  work  the  three  solutions  mentioned  are 
combined  in  equal  parts. 

Benedict's  Sugar  Reagent  (Method  No.  2).^ 

Copper  sulphate  (crj'stallized) 18.0  grams. 

Sodium  carbonate  (crystallized,  one-half  the  weight  of  the 

anhydrous  salt  may  be  used) 200 .0  grams. 

Sodium  or  potassium  citrate 200.0  grams. 

Potassium  thiocyanate 125 .0  grams. 

Potassium  ferrocyanide  (5  per  cent  solution) 5.0  c.c. 

Distilled  water  to  make  a  total  volume  of 1000. o  c.c. 

With  the  aid  of  heat  dissolve  the  carbonate,  citrate  and  thiocyanate 
in  enough  water  to  make  about  800  c.c.  of  the  mixture  and  filter  if  necessary. 

*  Quantitative  determination  of  sugar,  page  385. 


APPENDIX,  445 

Dissolve  the  copper  sulphate  separately  in  about  loo  c.c.  of  water 
and  pour  the  solution  slowly  into  the  other  liquid,  with  constant  stirring. 
Add  the  ferrocyanide  solution,  cool  and  dilute  to  exactly  i  liter.  Of  the 
various  constituents,  the  copper  salt  only  need  be  weighed  with  exactness. 
Twenty-live  cubic  centimeters  of  the  reagent  are  reduced  by  50  mg.  of 
glucose. 

Bial's  Reagent.'    • 

On  inol 1.5     grams. 

Fuming  HCl 500 .  00  grams. 

Ferric  chloride  (10  per  cent) 20-30     drops. 

Benedict's  Sulphur  Reagent. 

Crystallized  copper  nitrate,  sulphur-free  or  of  known  sulphur 

content 200  grams. 

Sodium  or  potassium  chlorate 50  grams. 

Distilled  water  to 1000   c.c. 

Black's  Reagent.- — Made  by  dissolving  5  grams  of  ferric  chloride 
and  0.4  gram  of  ferrous  chloride  in  100  c.c.  of  water. 

Blood  Serum. — This  may  easily  be  obtained  in  quantity  by  the 
procedure  described  under  Hemagglutination  in  the  chapter  on  Blood. 

Boas'  Reagent.^ — Dissolve  5  grams  of  rcsorcinol  and  3  grams  of 
sucrose  in  100  c.c.  of  50  per  cent  alcohol. 

Bonnano's  Reagent. — Dissolve  2  grams  of  sodium  nitrite  in  100 
c.c.  of  concentrated  hydrochloric  acid. 

Bottu's  Reagent. — To  3.5  grams  of  (7-nitrophenylpropiolic  acid 
add  5  c.c.  of  a  freshly  prepared  10  per  cent  solution  of  sodium  hydroxide 
and  make  the  volume  of  the  solution  one  liter  with  distilled  water. 

Combined  Hydrochloric  Acid  (Protein  Salt). — To  prepare  so- 
called  combined  hydrochloric  acid  simply  add  a  soluble  protein  such  as 
Witte's  peptone  to  free  hydrochloric  acid  of  the  desired  strength  until 
it  no  longer  responds  to  free  acid  tests  (see  chapter  on  Gastric  Digestion). 
For  example,  if  0.2  per  cent  combined  acid  is  required  the  protein  would 
be  added  to  0.2  per  cent  free  hydrochloric  acid. 

Strictly  speaking  there  is  no  such  thing  as  "combined"  acid  in  this 
sense.  When  the  protein  is  added  a  protein  salt  of  the  acid  is  formed  which 
ionizes  differently  from  the  free  acid. 

Congo  Red.^ — ^Dissolve  0.5  gram  of  congo  red  in  90  c.c.  of  water 
and  add  10  c.c.  of  95  per  cent  alcohol. 

Cross  and  Bevan's  Reagent. — Combine  tivo  parts  of  concentrated 
hydrochloric  acid  and  one  part  of  zinc  chloride  by  weight. 

'  Test  for  pentose,  page  ^^2. 

•  Black"*  reaction,  page  350. 

'  Test  for  free  acid,  page  130. 

*  Test  for  free  acid,  page  130. 


446  PHYSIOLOGICAL   CHEMISTRY. 

Ehrlich's  Diazo  Reagent.^— Two  separate  solutions  should  be 
prepared  and  mixed  in  definite  proportions  when  needed  for  use. 

{a)  Five  grams  of  sodium  nitrite  dissolved  in  i  liter  of  distilled  water. 

{b)  Five  grams  of  sulphanilic  acid  and  50  c.c.  of  hydrochloric  acid  in 
I  liter  of  distilled  water. 

Solutions  a  and  b  should  be  preserved  in  well-stoppered  vessels  and 
mixed  in  the  proportion  i :  50  when  required.  Green  asserts  that  greater 
delicacy  is  secured  by  mixing  the  solutions  in  the  proportion  i :  100. 
The  sodium  nitrite  deteriorates  upon  standing  and  becomes  unfit  for 
use  in  the  course  of  a  few  weeks. 

Esbach's  Reagent.^ — Dissolve  10  grams  of  picric  acid  and  20  grams 
of  citric  acid  in  i  liter  of  water. 

Fehling's  Solution.^ — ^Fehling's  solution  is  composed  of  two  definite 
solutions — a  copper  sulphate  solution  and  an  alkaline  tartrate  solution, 
which  may  be  prepared  as  follows : 

Copper  sulphate  solution  =  2)A-^S  grams  of  copper  sulphate  dissolved 
in  water  and  made  up  to  500  c.c. 

Alkaline  tartrate  solution  =12^  grams  of  potassium  hydroxide  and 
173  grams  of  Rochelle  salt  dissolved  in  water  and  made  up  to  500  c.c. 

These  solutions  should  be  preserved  separately  in  rubber-stoppered 
bottles  and  mixed  in  equal  volumes  when  needed  for  use.  This  is  done 
to  prevent  deterioration. 

Ferric  Alum  Solution/ — A  cold  saturated  solution. 

Folin-Shaffer  Reagent/ — This  reagent  consists  of  500  grams  of 
ammonium  sulphate,  5  grams  of  uranium  acetate,  and  60  c.c.  of  10  per 
cent  acetic  acid  in  650  c.c.  of  distilled  water. 

Furfurol  Solution/ — Add  i  c.c.  of  furfurol  to  1000  c.c.  of  distilled 
water. 

Gallic  Acid  Solution/ — A  saturated  alcoholic  solution. 

Gies'  Biuret  Reagent. — This  reagent  consists  of  10  per  cent  KOH 
solution  to  which  enough  3  per  cent  CuSO^  solution  has  been  added 
to  impart  a  slight  though  distinct  blue  color  to  the  clear  liquid.  The 
CuSO^  should  be  added  drop  by  drop  with  thorough  shaking  after 
each  addition. 

Guaiac  Solution/ — Dissolve  0.5  gram  of  guaiac  resin  in  30  c.c. 
of  95  per  cent  alcohol. 

'  Ehrlich's  diazo  reaction,  page  359. 

^  Esbach's  method,  page  383. 

'  Fehling's  method,  page  384.     Fehling's  test,  pages  32  and  327. 

*  Volhard-Arnold  method,  page  419. 
'  Folin-Shaffer  method,  page  389. 

"  Mylius's  modification  of  Pettenkofer's  test,  pages  164  and  344.  v.  Udransky's  test,  pages 
164  and  344. 

'  Gallic  acid  test,  page  243. 

*  Guaiac  test,  pages  186,  209  and  240. 


APPENDIX.  447 

Giinzberg's  Reagent/ — Dissolve  2  grams  of  phloroglucinol  and  i 
gram  of  vanillin  in  100  c.c.  of  95  per  cent  alcohol. 

Hammarsten's  Reagent." — Mix  i  volume  of  25  per  cent  nitric 
acid  and  19  volumes  of  25  per  cent  hydrochloric  acid  and  add  i  volume 
of  this  acid  mixture  to  4  volumes  of  95  per  cent  alcohol.  It  is  perfer- 
able  that  the  acid  mixture  be  prepared  in  advance  and  allowed  to  stand 
until  yellow  in.color  before  adding  it  to  the  alcohol. 

Hayem's  Solution. — This  solution  has  the  following  formula: 

Mcrcuri  ■  chlori  le o  25  grams. 

Sodium  chl  )ri  le 0.5  grams. 

Sodium  suli)liate 2.5  grams. 

DiililL'd  water loo.o  grams. 

Hopkins-Cole  Reagent.^ — To  one  liter  of  a  saturated  solution  of 
oxalic  acid  add  60  grams  of  sodium  amalgam  and  allow  the  mixture 
to  stand  until  the  evolution  of  gas  ceases.  Filter  the  dilute  with  2-3 
volumes  of  water. 

Hopkins-Cole  Reagent  (Benedict's  Modification). — Ten  grams 
of  powdered  magnesium  are  placed  in  a  large  Erlenmcyer  flask  and 
shaken  up  with  enough  distilled  water  to  liberally  cover  the  magnesium. 
Two  hundred  and  fifty  cubic  centimeters  of  a  cold,  saturated  solution 
of  oxalic  acid  is  now  added  slowly.  The  reaction  proceeds  very  rapidly 
and  with  the  liberation  of  much  heat,  so  that  the  flask  should  be  cooled 
under  running  water  during  the  addition  of  the  acid.  The  contents 
of  the  flask  arc  shaken  after  the  addition  of  the  last  portion  of  the  acid 
and  then  poured  upon  a  filter,  to  remove  the  insoluble  magnesium  oxalate. 
A  little  wash  water  is  poured  through  the  filter,  the  filtrate  acidified  with 
acetic  acid  to  prevent  the  partial  precipitation  of  the  magnesium  on  long 
standing,  and  made  up  to  a  liter  with  distilled  water.  This  solution 
contains  only  the  magnesium  salt  of  glyoxylic  acid. 

Hypobromite  Solution.^ — The  ingredients  of  this  solution  should 
be  prepared  in  the  form  of  two  separate  solutions  which  may  be  united 
as  needed. 

(a)  Dissolve  125  grams  of  sodium  bromide  in  water,  add  125  grams 
of  bromine  and  make  the  total  volume  of  the  solution  i  liter. 

{b)  A  solution  of  sodium  hydroxide  having  a  specific  gravity  of 
1.25.     This  is  approximately  a  22.5  per  cent  solution. 

Preserve  both  solutions  in  rubber-stoppered  bottles  and  when  needed 
for  use  mix  one  volume  of  solution  a,  one  volume  of  solution  b,  and  3 
volumes  of  water. 

'  Test  for  free  acid,  page  130. 

^  Hammarsten's  reaction,  pages  163  and  343. 

'  Hopkins-Cole  reaction,  page  q8. 

*  Methods  for  determination  of  urea,  page  392. 


448  PHYSIOLOGICAL    CHEMISTRY, 

Iodine  Solution.^ — Prepare  a  2  per  cent  solution  of  potassium 
iodide  and  add  sufficient  iodine  to  color  it  a  deep  yellow. 

Iodine-Zinc  Chloride  Reagent.- — Dissolve  20  grams  of  zinc  chloride 
in  8.5  c.c.  of  water.  Cool,  and  introduce  iodine  solution  (3  grams  KI  + 
1.5  gram  I  in  60  c.c.  of  water)  drop  by  drop  until  iodine  begins  to 
precipitate. 

JoUes'    Reagent.^ — This   reagent   has   the   following   composition: 

Succinic  acid 40  grams. 

Mercuric  chloride 20  grams. 

Sodium  chloride 20  grams. 

Distilled  water 1000  grams. 

Kantor  and  Gies'  Biuret  Paper.* — Immerse  filter  paper  in  Gies' 
Biuret  Reagent  (p.  99)  then  dry  and  cut  into  strips. 

Kraut's  Reagent.^ — Dissolve  272  grams  of  potassium  iodide  in 
water  and  add  80  grams  of  bismuth  subnitrate  dissolved  in  200  grams 
of  nitric  acid  (sp.  gr.  1.18).  Permit  the  potassium  nitrate  to  crystallize 
out,  then  filter  it  off  and  make  the  filtrate  up  to  i  liter  with  water. 

Lugol's  Solution.*' — Dissolve  4  grams  of  iodine  and  6  grams  of 
potassium  iodide  in  100  c.c.  of  distilled  water. 

Magnesia  Mixture.' — Dissolve  175  grams  of  magnesium  sulphate 
and  350  grams  of  ammonium  chloride  in  1400  c.c.  of  distilled  water. 
Add  700  grams  of  concentrated  ammonium  hydroxide,  mix  thoroughly, 
and  preserve  the  mixture  in  a  glass-stoppered  bottle. 

Millon's  Reagent.^ — Digest  i  part  (by  weight)  of  mercury  with 
2  parts  (by  weight)  of  nitric  acid  (sp.  gr.  1.42)  and  dilute  the  resulting 
solution  with  2  volumes  of  water. 

Molisch's  Reagent.''' — ^A  15  per  cent  alcoholic  solution  of  a-naphthol. 

Molybdic  Solution.^" — Molybdic  solution  is  prepared  as  follows, 
the  parts  being  by  weight 

Molybdic  add i   part. 

Ammonium  hydroxide  (sp.  gr.  o .  96) 4  parts. 

Nitric  acid  (sp.  gr.  1.2) 15  parts. 

Moreigne's  Reagent.^* — Combine  20  grams  of  sodium  tungstate, 

'  Iodine  test,  page  50. 

^  .A.myloid  formation,  p.  54. 

'  Jolles'  reaction,  pages  105  and  334. 

*  Protein  tests,  p.  332. 

*  Rosenheim's  bismuth  test  for  choline,  page  273. 

"  Gunning's  iodoform  test,  page  346,  and  Bardach's  reaction,  page  loi. 

'  Sodium  hydroxide  and  potassium  nitrate  fusion  method  for  determination  of  total  phos- 
phorus, page  414. 

"  Millon's  reaction,  page  97. 

■  Molisch's  reaction,  page  27. 

'"  Sodium  hydroxide  and  potassium  nitrate  fusion  method  for  determination  of  total  phos- 
phorus, page  414. 

' '  Moreigne's  reaction,  page  293. 


■APPENDIX.  449 

lo  grams  of  phosphoric  acid  (sp.  gr.  1.13)  and  100  c.c.  of  water.  Boil 
the  mixture  for  twenty  minutes,  add  water  to  make  the  volume  of  the 
solution  equivalent  to  the  original  volume,  and  acidify  with  hydrochloric 
acid. 

Morner's  Reagent.'— Thoroughly  mix  i  volume  of  formalin,  45 
volumes  of  distilled  water,  and  55  volumes  of  concentrated  sulphuric 
acid. 

Nakayama's  Reagent.-- — Prepared  by  combining  99  c.c.  of  alcohol 
and  I  c.c.  of  fuming  hydrochloric  acid  containing  4  grams  of  ferric 
chloride  per  liter. 

Nessler-Winkler  Solution. 

Mercuric  iodide .' lo  grams. 

Potassium  iodide 3   grams. 

Sodium  hydroxide 20  grams. 

Water 100  c.c. 

The  mercuric  iodide  is  rubbed  up  in  a  small  porcelain  mortar  with 
water,  then  washed  into  a  tlask  and  the  potassium  iodide  added.  The 
sodium  hydroxide  is  dissolved  in  the  remaining  water  and  the  cooled  solu- 
tion added  to  the  above  mixture.  The  solution  cleared  by  standing  is 
preserved  in  a  dark  bottle. 

Neutral  Olive  Oil.^ — Shake  ordinary  olive  oil  w^ith  a  lo  per  cent 
solution  of  sodium  carbonate,  extract  the  mixture  with  ether,  and  remove 
the  ether  by  evaporation.     The  residue  is  neutral  olive  oil. 

Nylander's  Reagent.'* — Digest  2  grams  of  bismuth  subnitrate 
and  4  grams  of  Rochelle  salt  in  100  c.c.  of  a  10  per  cent  solution  of  potas- 
sium hydroxide.     The  reagent  should  then  be  cooled  and  filtered. 

Obermayer's  Reagent.^ — Add  2-4  grams  of  ferric  chloride  to  a 
liter  of  hydrochloric  acid  (sp.  gr.  1.19). 

Oxalated  Plasma.^ — Allow  arterial  blood  to  run  into  an  equal 
volume  of  0.2  per  cent  ammonium  oxalate  solution. 

Para-dimethylaminobenzaldehyde  Solution.'' — This  solution  is 
made  by  dissolving  5  grams  of  para-dimethylaminobenzaldehyde  in 
100  c.c.  of  10  per  cent  sulphuric  acid. 

Para-phenylenediamine  Hydrochloride  Solution.^ — Two  grams 
dissolved  in  too  c.c.  of  water. 

Phenolphthalein.® — Dissolve  i  gram  of  phenolphthalein  in  100 
c.c.  of  95  per  cent  alcohol. 

'  Morner's  test,  page  91. 

'Nakayama's  reaction,  pages  162  and  342. 

*  Emulsification  of  fats,  page  143. 

*  Nylander's  test,  pages  34  and  330. 

*  Obermayer's  test,  page  299. 

*  Experiments  on  blood  plasma,  page  214. 

^  Herter's  para-dimethylaminobenzaldehyde  reaction,  page  176. 

*  Detection  of  hydrogen  peroxide,  page  244. 
'  Topfer's  method,  page  440. 

29 


450  PHYSIOLOGICAL   CHEMISTRY. 

Phenylhydrazine  Mixture/ — This  mixture  is  prepared  by  com- 
bining I  part  of  phenylhydrazine-hydrochloride  and  2  parts  of  sodium 
acetate  by  weight.     These  are  thoroughly  mixed  in  a  mortar. 

Phenylhydrazine-acetate  Solution.- — This  solution  is  prepared 
by  mixing  i  volume  of  glacial  acetic  acid,  i  volume  of  water,  and  2 
volumes  of  phenylhydrazine  (the  base). 

Purdy's  Solution.^ — Purdy's  solution  has  the  following  composition: 

Copper  sulphate    4-752  grams. 

Potassium  hydroxide    23.5  grams. 

Ammonia   (U.  S.  P.,  sp.  gr.  o.  9)      35° •  o  c.c. 

Glycerol    38 .  o  c.c. 

Distilled  water,  to  make  total  volume  i  liter. 

Roberts'  Reagent/ — Mix  i  volume  of  concentrated  nitric  acid 
and  5  volumes  of  a  saturated  solution  of  magnesium  sulphate. 

Rosenheim's  lodo-Potassium  Iodide  Solution/ — Dissolve  2  grams 
of  iodine  and  6  grams  of  potassium  iodide  in  100  c.c.  of  water. 

Salted  Plasma/' — Allow  arterial  blood  to  run  into  an  equal  vol- 
ume of  a  saturated  solution  of  sodium  sulphate  or  a  10  per  cent  solu- 
tion of  sodium  chloride.  Keep  the  mixture  in  the  cold  room  for  about 
24  hours. 

Schiff's  Reagent/ — This  reagent  consists  of  a  mixture  of  three 
volumes  of  concentrated  sulphuric  acid  and  one  volume  of  10  per  cent 
ferric  chloride. 

Schweitzer's  Reagent/ — Add  potassium  hydroxide  to  a  solution 
of  copper  sulphate  which  contains  some  ammonium  chloride.  Filter 
off  the  precipitate  of  cupric  hydroxide,  wash  it,  and  bring  3  grams  of 
the  moist  cupric  hydroxide  into  solution  in  a  liter  of  20  per  cent  ammo- 
nium hydroxide. 

Seliwanoff's  Reagent/ — Dissolve  0.05  gram  of  resorcinol  in  100 
c.c.  of  dilute  (1:2)  hydrochloric  acid. 

Sherrington's  Solution/*^ — This  solution  possesses  the  following 
formula: 

Methylene-blue o .  i   gram. 

Sodium  chlorirle 12    gram. 

Neutral  potassium  o.xalate 1.2   gram. 

Distilled  water 300 .0  grams. 

Sodium  Acetate  Solution/^ — Dissolve  loo  grams  of  sodium  acetate 

'  Phenylhydrazine  reaction,  pages  28  and  324. 

-  Phenylhydrazine  reactiDn,  pa^es  28  and  324. 

^  Purdy's  method,  page  387. 

■*  Roberts'  ring  test,  pages  104  and  334. 

^  Rosenheim's  periorlide  test,  page  273. 

"Experiments  on  blood  plasma,  page  214. 

^  Schiff's  reaction,  pages  i66  and  272. 

*  Schweitzer's  solubility  test,  page  54. 

*  Seliwanoff's  reaction,  pages  40  and  356. 
'""Blood  counting,"  page  224. 

"  Uranium  acetate  method,  page  413. 


APPENDDC.  451 

in  800  c.c.  of  distilled  water,  add  too  c.c.  of  30  per  cent  acetic  acid  to  the 
solution,  and  make  the  volume  of  the  mixture  up  to  i  liter  with  distilled 
water. 

Sodium  Alizarin  Sulphonate.' — Dissolve  i  gram  of  sodium  aliz- 
arin sulphonate  in  100  c.c.  of  water. 

Sodium  Sulphide  Solution.- — Saturate  a  i  per  cent  solution  of 
sodium  hydcoxide  with  hydrogen  sulphide  gas  and  add  an  equal  volume 
of  I  per  cent  sodium  hydroxide. 

Solera's  Test  Paper. ^ — Saturate  a  good  cjuality  of  filter  paper 
with  0.5  per  cent  starch  paste  to  which  has  been  added  sufficient  iodic 
acid  to  make  a  i  per  cent  solution  of  iodic  acid  and  allow  the  paper  to 
dry  in  the  air.     Cut  it  in  strips  of  suitable  size  and  preserve  for  use. 

Spiegler's  Reagent.'' — This  reagent  has  the  following  composition: 

Tartaric  acid 20  grams. 

Mercuric  chloride 40  grams. 

Glycerol 100  grams. 

Distilled  water 1000  grams. 

Standard  Ammonium  Thiocyanate  Solution.^ — This  solution  is 
made  of  such  a  strength  that  i  c.c.  of  it  is  equal  to  i  c.c.  of  the  standard 
silver  nitrate  solution  mentioned  below.  To  prepare  the  solution  dissolve 
12.9  grams  of  ammonium  thiocyanate,  NH^SCN,  in  a  little  less  than  a 
liter  of  water.  In  a  small  flask  place  20  c.c.  of  the  standard  silver  nitrate 
solution,  5  c.c.  of  a  cold  saturated  solution  of  ferric  alum  and  4  c.c.  of 
nitric  acid  (sp.  gr.  1.2),  add  water  to  make  the  total  volume  100  c.c,  and 
thoroughly  mix  the  contents  of  the  flask.  Now  run  in  the  ammonium 
thiocyanate  solution  from  a  burette  until  a  permanent  red-brown  tinge  is 
produced.  This  is  the  end-reaction  and  indicates  that  the  last  trace 
of  silver  nitrate  has  been  precipitated.  Take  the  burette  reading  and 
calculate  the  amount  of  water  necessary  to  use  in  diluting  the  ammonium 
thiocyanate  in  order  that  10  c.c.  of  this  solution  may  be  exactly  equal 
to  10  c.c.  of  the  silver  nitrate  solution.  Make  the  dilution  and  titrate 
again  to  be  certain  that  the  solution  is  of  the  proper  strength. 

Standard  Silver  Nitrate  Solution." — Dissolve  29.042  grams  of 
silver  nitrate  in  i  liter  of  distilled  water.  Each  cubic  centimeter  of  this 
solution  is  equivalent  to  0.0 1  gram  of  sodium  chloride  or  to  0.006  gram 
of  chlorine. 

Standard  Uranium  Acetate  Solution.^ — Dissolve  about  34  grams  of 

'  Topfer's  method,  page  440. 

^  Kniger  and  Schmidt's  method,  pages  391  and  429. 

'  Solera's  reaction,  page  64. 

*  Spiegler's  ring  test,  pages  104  and  334. 

*  Volhard-.Arnold  method,  page  419,  and  Dehn-Clark  method,  page  417. 

*  Volhard- Arnold  method,  page  419,  Mohr's  method,  page  418,  and  Dehn-Clark  method, 
page  417. 

'  Uranium  acetate  method,  page  413. 


452  PHYSIOLOGICAL   CHEMISTRY. 

uranium  acetate  in  i  liter  of  water.  One  c.c.  of  such  a  solution  should  now 
be  made  equivalent  to  0.005  gram  of  PzOg,  phosphoric  anhydride.  It 
may  be  standardized  as  follows:  To  50  c.c.  of  a  standard  solution  of 
disodium  hydrogen  phosphate,  of  such  a  strength  that  the  50  c.c. 
contains  o.i  gram  of  PgOg,  add  5  c.c.  of  the  sodium  acetate  solution 
mentioned  on  p.  450  and  titrate  with  the  uranium  solution  to  the  correct 
end-reaction  as  indicated  in  the  method  proper  on  p.  413.  Inasmuch  as 
I  c.c.  of  the  uranium  solution  should  precipitate  0.005  gram  of  PjOj, 
exactly  20  c.c.  of  the  uranium  solution  should  be  required  to  precipitate 
the  50  c.c.  of  the  standard  phosphate  solution.  If  the  two  solutions  do 
not  bear  this  relation  to  each  other  they  must  be  brought  into  the  proper 
relation  by  diluting  the  uranium  solution  with  distilled  water  or  by  in- 
creasing its  strength. 

Starch  Iodide  Solution/ — Mix  o.i  gram  of  starch  powder  with 
cold  water  in  a  mortar  and  pour  the  suspended  starch  granules  into  75-100 
c.c.  of  boiling  water,  stirring  continuously.  Cool  the  starch  paste,  add 
20-25  grams  of  potassium  iodide  and  dilute  the  mixture  to  50  c.c.  This 
solution  deteriorates  upon  standing,  and  therefore  must  be  freshly  pre- 
pared as  needed. 

Starch  Paste. ^ — Grind  2  grams  of  starch  powder  in  a  mortar  with  a 
small  amount  of  water.  Bring  200  c.c.  of  water  to  the  boiling-point  and 
add  the  starch  mixture  from  the  mortar  with  continuous  stirring.  Bring 
again  to  the  boiling-point  and  allow  it  to  cool.  This  makes  an  approxi- 
mate I  per  cent  starch  paste  which  is  a  very  satisfactory  strength  for 
general  use. 

Stokes'  Reagent.^ — ^A  solution  containing  2  per  cent  ferrous  sulphate 
and  3  per  cent  tartaric  acid.  When  needed  for  use  a  small  amount  should 
be  placed  in  a  test-tube  and  ammonium  hydroxide  added  until  the 
precipitate  which  forms  on  the  first  addition  of  the  hydroxide  has  entirely 
dissolved.  This  produces  ammonium  ferrotartrate  which  is  a  reducing 
agent. 

Suspension  of  Manganese  Dioxide.^ — Made  by  heating  a  0.5  per 
cent  solution  of  potassium  permanganate  with  a  little  alcohol  until  it  is 
decolorized. 

Tanret's  Reagent.^ — Dissolve  1.35  grams  of  mercuric  chloride  in 
25  c.c.  of  water,  add  to  this  solution  3.32  grams  of  potassium  iodide 
dissolved  in  25  c.c.  of  water,  then  make  the  total  solution  up  to  60  c.c. 
with  distilled  water  and  add  20  c.c.  of  glacial  acetic  acid  to  the  mixture. 

'  Fehling's  method,  page  384. 
-  Fehling's  method,  page  384. 
'  HLaemoglobin,  page  216.     Haemochromogen,  page  219. 

*  Kriiger  and  Schmidt's  method,  pages  391  and  429. 

*  Tanret's  test,  pages  104  and  334. 


APPENDIX.  453 

Tincture  of  Iodine.* — Dissolve  70  grams  of  iodine  and  50  grams  of 
potassium  iodide  in  i  liter  of  95  per  cent  alcohol. 

Toison's  Solution.^ — This  solution  has  the  following  formula: 

Methyl  violet o  .025  gram. 

Sodium  chloride : i  .0  gram. 

Sodium  sulphate 8.0  grams. 

Glycerol 30 .0  grams. 

Distilled  water 160.0  grams. 

Topfer's  Reagent.^ — Dissolve  0.5  gram  of  di-methylaminoazobenzene 
in   100  c.c.  of  95  per  cent  alcohol. 

Tropaeolin  00.^ — Dissolve  0.05  gram  of  tropasolin  00  in  100  c.c. 
of  50  per  cent  alcohol. 

Ufifelmann's  Reagent.^ — Add  a  5  per  cent  solution  of  ferric  chloride 
to  a  I  per  cent  solution  of  carbolic  acid  until  an  amethyst-blue  color  is 
obtained. 

'  Smith's  test,  pages  163  and  343. 
^  "  Blood  counting,"  page  224. 
'  Topfer's  method,  page  440. 

*  Test  for  free  acid,  page  130. 

*  Uffelmann's  reaction,  page  136. 


NDEX. 


Acacia  solution,  fwrmation  of  emulsion  by,  141 
Acetone,  323,  345 

formula  for,  ,14s 

Gunning's  iodoform  test  for,  346 

Legal's  iodoform  test  for,  346 

Lieben's  test  for,  347 

quantitative  determination  of,  423 

Reynolds-Gunning  test  for,  347 

Rothera's  reaction  for,  347 

Taylor's  test  for,  347 
Acholic  stool,  179 
Achroo-dextrins,  48,  61,  65 

a-achroo-dextrin,  61 

^-achroo-dextrin,  61 

r-achroo-dextrin,  61 
Acid,  acetic,  284,  309 

alloxyproteic,  283,  303,  359 

amino-acetic,  58,  74,  77 

amino-butyric,  171 

amino-ethyl-sulphonic,  159,  260 

a-amino-;9-hydroxy-propionic,  74.  78 

a-amino-/3-imidazol-propionic,  73,  82 

o-amino-iso-butyl-acetic,  74,  84 

a-amino-;S-methyl  ,3-ethyl-propionic,  73,  85 

a-amino-normal  glutaric,  74,  87 

o-amino-propionic,  73,  77 

amino-succinic,  74,  86 

amino- valerianic,  171 

a-amino-iso-valerianic  (see  Valine),  74,  83, 

a-diamino-^-dithiolactyl  73,  80 

aspartic,  74,  86 

benzoic,  168,  283,  307 

butyric,  8,  238,  243,  284,  309 

caproic,  235,  238 

carbamic,  196 

cholic,  159 

chondroitin-sulphuric.  250,  283,  303 

citric,  23s 

combined  hydrochloric  (protein  salt),  61,  126, 
441 

cyanuric,  286 

a-e-di-amino-caproic,  73,  85 

diaminotrihydroxydodecanoic,  73,  89 

diazo-benzene-sulphonic,  360 

ethereal  sulphuric,  169,  283,  297 

fatty,  139,  140,  14s,  284,  383 

formic,  284,  309 

free  hydrochloric,  61,  66,  130,  441 

glucothionic,  371 

glutamic,  74,  87 

glycocholic,  159 

glycosuric,  306 

glycuronic,  42,  353 

glycerophosphoric,  268,  269,  284.  3 to 

glyoxylic,  97,  98 

guanidine-a-amino- valerianic,  73,  83 

hippuric,  168,  283,  300,  406 


Acid,  homogentisic,  32,  283,  306,  327 
iniinazalpropionic,  171 
indolacetic,  171,407 
indole- rt-amino-propionic,  73,  82 
indoxyl-sulphuric,  169,  297 
inosinic,  25s,  260 
kynurenic,  283,  307 
lactic,  45,  126,  136,  138,  235,  256 
lauric,  235 

mucic,  41,  45,  354,  .355 
myristic,  235 
nucleic,  94,  112 
osmic,  271,  299 
oxalic,  283,  302 
oxaluric,  283,  308 

oxy-a-pyrrolidine-carboxylic,  73,  89 
oxymandelic,  283,  306 
oxyproteic,  283,  303,  359 
palmitic,  140,  145,  151. 
para-cresol-sulphuric,  283,  297 
para-oxyphenyl-aceti(i    169,    171,    17s.  283, 

303 
para-oxy-;3-phenyl-a-amino-propionic,  74,  79 

para-oxyphenyl-propionic,     169,     171,     I7  5i 

283,  303 
paralactic,  23s,  256,  284,  309 
phenaceturic,  284,  309.  4°? 
phenol-sulphuric,  283,  297 
phenylacetic,  171 
phenyl- a-amino  propionic,  74,  78 
phenylpropionic,  171 
phosphocamic,  25s,  260,  284,  310 
phosphoric,  3  17 

pyrocatechin-sulphuric,  283,  297 
a-pyrrolidine-carboxylic,  73,  88 
sarcolactic,  256 
skatole  acetic,  82 
skatole-carbonic,  174,  175 
skatoxyl-sulphuric,  283,  297 
stearic,  269 
succinic,  i  7  i 
sulphanilic,  360 
tannic,  50,  53 
taurocholic,  159 

uric,  32.  255,  277,  283,  290,  362,  368 
uroferric,  283,  303.  359 
uroleucic,  283 

volatile  fatty,  169,  172,  284,  309 
Acid  albuminate.     See  Acid  metaprotein. 
Acid  infraprotein.     See  Acid  metaprotein. 
Acid  metaprotein,  1 16 
coagulation  of,  1 1 6 
experiments  on,  116 
precipitation  of,  116 
preparation  of,  116 
solubility  of,  116 
sulphur  content  of,  116 


455 


456 


INDEX. 


Acidity  of  gastric  juice,  quantitative  determina- 
tion of,  440 
urine,  cause  of,  276,  317 

quantitative  determination  of,  427 
Acidosis,  cause  of,  350 
Acid-haematin,  219 

Acree-Rosenheim  formaldehyde  reaction,  100 
Acrolein,  formation  of,  from  olive  oil,  143 

from  glycerol,  146 
Activation,  6,  150 
Activation  by  calcium  salts,  150 
^dam's  paper  coil  method  for  determination  of 

fat  in  milk,  43  7 
Adamkiewicz  reaction,  97 
Adaptation,  62 
Adenase,  4 

Adenine,  4,  261,  284,  312 
Adipocere,  142 
Adler's   benzidine   reaction   for   blood,    i8s,    204, 

209,  341 
Agar-agar,  26,  55,  56,  180 
Agglutination,  197 
Alanine,  73,  77,  171 
Albumin,  egg,  107 

powdered,  preparation  of,  107 

tests  on,  107 
serum,  93,  95,  194,  323,  332 
Albumin  in  urine,  323,  332 

acetic  acid  and  potassium  ferrocyanide 
test  for,  33  5 
coagulation  or  boiling  test  for,  33s 
Heller's  ring  test  for,  333 
JoUes'  reaction  for,  334 
Roberts'  ring  test  for,  334 
sodium  chloride  and  acetic  acid  test  for,  336 
Spiegler's  ring  test  for,  334 
Tanret's  test  for,  335 
tests  for,  333 
Albumins,  93,  95,  96 
Albuminates.     See  Metaproteins. 
Albuminates,    formation    of,    by    metallic    salts, 

102,  103 
Albuminoids,  93,  112 
Albumoscope,  104,  334 
Albumoses  (see  Proteoses,  p.  119) 
Alcohol-soluble  proteins.     See  Prolamins. 
Aldehyde,  25,  30 
Aldehyde  group,  44 
Aldehyde  test  for  alcohol,  47 
v.    Aldor's    method    of    detecting    proteose    in 

urine,  338 
Aldose,  25 

Aliphatic  nucleus,  73,  74 
Alkali  albuminate.     See  Alkali  metaprotein. 
Alkali-hffimatin,  212,  219 
Alkali  metaprotein,  94,  116,  117 
experiments  on,  117 
precipitation  of,  117 
preparation  of,  117 
sulphur  content  of,  117 
Alkaline  tide,  276 
AUantoin,  283,  303 

crystalline  form  of,  304 
experiments  on,  305 
formula  for,  303  ' 

preparation  of,  from  uric  acid,  305 
quantitative  determination  of,  432 
separation  of,  from  urine,  305 


Allen's  modification  of  Fehling's  test,  329 
Almen's  reagent,  preparation  of,  339 
Alloxyproteic  acid,  283,  303,  359 
Aloin -turpentine   test    for    "occult    blood,"    181, 

i8s 
Amandin,  93 
Amide  nitrogen,  69 

Amidulin.     See  Soluble  starch,  18,  48,  6i 
Amino  acids,  69,  93,  149,  159,  171,  283 

group,  95,  98 
a-amino-^-hydroxy-propionic  acid,  74,  78 
a-amino-/3-imidazol-propionic  acid,  73,  82 
a-amino-iso-butyl-acetic  acid,  74,  84 
a-amino-normal-glutaric  acid,  74,  87 

404 
Amino-butyric  acid,  171 

Amino-nitrogen,  quantitative    determination    of, 
Amino-succinic  acid,  74,  86 
Amino-valerianic  acid,  171 
a-amino-iso-valerianic  acid,  74,  83 
Ammonia,  69,  76,  108 
Ammonia  in  urine,  284,  313 

quantitative  determination  of,  399 
Ammoniacal  silver  solution,  preparation  of,  430 
Ammoniacal-zinc  chloride  test  for  urobilin,  311 
Ammonium     magnesium     phosphate      ("  Triple 
phosphate"),  277,  319 
in  urinary  sediments,  362 
Ammonium  urate,  290,  36s,  389 

crystalline  form  of,  Plate  VI,  opposite 
P-,  36s 
Amphopeptone,  95,  120 
Amylase,  pancreatic,  4,  10,  150 

digestion  of  dry  starch  by,  151,  156 

inulin  by,  157 
experiments  on,  10,  155 
influence  of  bile  upon  action  of,  156 

metallic  salts,  upon  action  of,  156 
most   favorable   temperature   for  action   of, 

iS6 
salivary,  4,  10,  60,  126 

activity  of,  in  stomach,  61,  126 
experiments  on,  10,  65 
inhibition  of  activity  of,  61,  66 
nature  of  action  of,  61 
products  of  action  of,  61 
vegetable,  4,  10 
Amylases,  3,  4,  10,  60,  150 

experiments  on,  10,  6s,  iSS 
Amyloid,  54,  113 
Amylolytic  enzymes.     See  Amylases. 

quantitative  determination   of  activity 
of,  18 
Animal  parasites  in  feces,  181,  183,  184 
in  urinary  sediments,  369,  378 
Antialbumid,  130 
Antienzymes,  9 

experiments  on,  17 
Antimony    pentachloride    as    cellulose    solvent, 

SS 
Antimony  trichloride  as  cellulose  solvent,  ss 
Antipepsin,  9,  17 
Antipeptone,  120 
Antirennin,  9 
Antithrombin,  204 
antitrypsin,  9,  18 
Aporrhegmas,  72,  170 
Appendix,  443 


INDEX. 


457 


Arabinose,  25,  42,  352 

Bial's  reaction  for,  42,  352 

orcinol  test  on,  43,  353 

phenylhydrazine  test  on,  43 

ToUens'  reaction  on,  42,  353 
Arginase,  4 

Arginine,  4,  72,  73.  83,  i49 
Amold-Lipliawsky  reaction  for  diacetic  acid,  349 

reagent,  preparation  of,  349 
Aromatic  oxyacids,  283,  306 
Ascaris,  17,18 
Asparagine,  86 
Aspartic  acid,  69,  72.  74.  86 

crystalline  form  of,  86 
formula  for,  86 
Ash  of  milk,  quantitative  determination  of,  438 
Assimilation  limit,  27,  41 
Assimilation  limit  of  dextrose,  27,  324 

galactose,  41 
Atkinson  and  Kendall's  haemin  test,  210 
Autol>-tic  enzymes,  3 

Babcock   fat  method,  435 

tube,  435 
Bacteria  in  feces,  181 

quantitative  determination  of,  191 
Barberio's  reaction  for  indican,  300 
Bardach's  reaction,  loi 

Barfoed's  reagent,  preparation  of,  14,  36,  331 
Barfoed's  test  for  monosaccharides,  36,  331 
Baryta  mixture,  preparation  of,  287 
Basic  lead  acetate  solution,  416,  443 
Bayberry  tallow,  saponification  of,  144 

source  of,  144 
Bayberry  wax.     See  Bayberry  tallow,  144 
Bead  test  (Einhom),  189 
Beckmann-Heidenhain  apparatus,  280 
"  Bence  Jones'  protein,"  detection  of,  338 
Benedict's     methods     for     quantitative     deter- 
mination of  sugar,  38s 
Benedict's     method      for      quantitative     deter- 
mination of  sulphur,  409 
Benedict's      method      for     quantitative      deter- 
mination of  urea,  396 
Benedict's  modifications  of  Fehling's  test,  33,  328 
solutions,  preparation  of,  33,  328 
solution,     for    use    in     quantitative    deter- 
mination of  sugar,  preparation  of,  385 
sulphur  reagent,  preparation  of,  409 
Benzidine  reaction,  Adler's,  for  blood,  185,  204, 

209,  341 
Benzoic  acid,  168,  283,  307 

crystalline  form  of,  308 
experiments  upon,  307 
formula  for,  307 
solubility  of,  307 
sublimation  of,  307 
Berthelot-Atwater  bomb  calorimeter,  411 
Bergell's    method   for  determination    of    ,3-oxy- 

butyric  acid,  427 
Bial's  reaction  for  pentoses,  42,  352 
Bial's  reagent,  preparation  of,  42,  352 
Bile,  158,  323,  342 

constituents  of,  159 
daily  secretion  of,  158 
freezing-point  of,  159 
influence  on  digestion,  gastric,  136 
pancreatic,  151,  155,  156 


Bile,   inorganic  constituents  of,  159,  162 
nucleoprotein  of,  162 
reaction  of,  158,  162 
secretion  of,  158 
specific  gravity  of,  159 
Bile  acids,  159 

Guerin's  reaction  for,  164 
Hay's  test  for,  164 
Mylius's  test  for,  164 
Neukomm's  test  for,  164 
Pettenkofer's  test  for,  163 
tests  for,  163 

V.  Udransky's  test  for,  164 
Bile  acids  in  feces,  detection  of,  187 
Bile  acids  in  urine,  323,  ^44 

Hay's  test  for,  344 
Mylius's  test  for,  344 
Neukomm's  test  for,  344 
Pettenkofer's  test  for,  344 
tests  for,  344 

V.  Udransky's  test  for,  344 
Bile  pigments,  160 

Gmelin's  test  for,  162 
Hammarsten's  reaction  for,  163 
Huppert's  reaction  for,  162 
Rosenbach's  test  for,  162 
Smith's  test  for,  163 
tests  for,  162 
Bile  pigments  in  urine,  323,  342 

Gmelin's  test  for,  342 
Hammarsten's  reaction  for,  343 
Huppert's  reaction  for,  342 
Nakayama's  reaction  for,  342 
Rosenbach's  test  for,  342 
Salkowski's  test  for,  343 
Salkowski-Schipper's   reaction    for, 

343 
Smith's  test  for,  343 
tests  for,  342 
Bile  salts,  7,  159 

crystallization  of,  159,  164 
Biliary  calculi,  161 

analysis  of,  165 
Bilicyanin,  160 
Bilifuscin,  160 
Bilihumin,  160 
Biliprasin,  160 
Bilirubin,  160 

crystalline  form  of,  161 
in  urinary  sediments,  362,  367 
Biliverdin,  160,  i6i 
"  Biological"  blood  test,  205 
Bismuth  test  for  choline,  273 
Biuret,  99,  286 

formation  of,  from  urea,  99,  286 
Biuret  paper  of  Kantor  and  Gies,  99 
Biuret  potassium  cupric  hydroxide.     See  Cupri- 
potassium  biuret,  99 
test,  98 

Posner's  modification  of,  100 
Biuret  reagent  (Gies),  preparation  of,  99 
Black's    method    for    determination    of    fi-O'x.y- 
butyric  acid,  426 
reaction  for  5-oxybutyric  acid,  350 
reagent,  preparation  of,  350 
Blood,  194.  323,  339 

agglutination  of,  197 
Bordet  test  for,  205 


458 


INDEX. 


Blood,  clinical  examination  of,  220 
coagulation  of,  195,  203 

Howell's  theory  of,  203 
constituents  of,  194,  196 
defibrinated,  206 
detection  of,  204,  209,  215 
erythrocytes  of,  194,  197,  212 
experiments  on,  206 
form  elements  of,  194 
guaiac  test  for,  186,  204,  209 
haemin  test  for,  204,  210 
"occult,"  in  feces,  181,  185 
oxyhemoglobin  of,  197,  216 
in  urine,  283,  339 
leucocytes  of,  202 
medico-legal  tests  for,  204 
microscopical  examination  of,  196,  204,  206, 

nucleoprotein  of,  194,  195 
pigment  of,  197 
plaques,  203 
plasma,  194,  214 
plates,  203 
platelets,  203 

preparation  of  haematin  from,  212 
preparation  of  "laky,"  207 
quantitative  analysis  of,  220 
reaction  of,  194,  206 
serum,  195,  213 
specific  gravity  of,  194,  206 
spectroscopic  examination  of,  215 
test  for  iron  in,  207 
total  amount  of,  194 

V.  Zeynek  and  Nencki's  hsemin  test  for,  210 
Blood  casts  in  urine,  369,  373 
Blood  corpuscles,  194,  196,  202 

"counting,"  224,  228 
Blood  dust,  194,  203 
Blood  in  urine,  323,  339 

Adler's  benzidine  reaction  for,  341 
guaiac  test  for,  341 
Teichmann's  hsemin  test  for,  340 
Heller's  test  for,  340 
Heller-Teichmann  reaction  for,  340 
Schalfijew's  hsemin  test  for,  340 
Schumm's    modification  of  guaiac  test 

for,  342 
spectroscopic  examination  of,  342 
tests  for,  340 

V.  Zeynek  and  Nencki's  haemin  test  for, 
340 
Blood  plasma,  194,  214 

constituents  of,  194 

crystallization     of    oxyhsemoglobin     of, 

201,  214 
effect  of  calcium  on  oxalated,  214 
experiments  on,  214 
preparation  of  fibrinogen  from,  214 
oxalated,  214 
salted,  214 
Blood  serum,  195,  213 

coagulation  temperature  of,  213 
constituents  of,  195 
experiments  on,  213 
precipitation  of  proteins  of,  213 
separation   of  albumin  and  globulin  of, 

2'.? 

sodium  chloride  in,  213 


Blood  serum,  sugar  in,  213 

Blood  stains,  examination  of,  215 

Boas'  reagent,  as  indicator,  132 
preparation  of,  132 

Boekelman    and     Bouma's     method    for    deter- 
mination of  /?-oxybutyric  acid,  427 

Boettger's  test  for  sugar,  34,  330 

Bomb  calorimeter,  Berthelot-Atwater,  411 

Bonanno's  reaction,  163,  343 

Bonanno's  reagent,  preparation  of,  163,  343 

Bone,  constituents  of,  251 

ossein  of,  preparation  of,  251 
quantitative  composition  of,  252 

Bone  ash,  scheme  for  analysis  of,  253 

Borchardt's  reaction  for  laevulose,  40,  356 

Bordet  test,  detection  of  human  blood  by,  205 

Boric  acid  and  borates  in  milk,  detection  of,  244 

Bottu's  reagent,  preparation  of,  29,  325 

Bottu's  test,  29,  325 

Bromelin,  4 

Buccal  glands,  59 

Buffy  coat,  formation  of,  196 

Bunge's  mass  action  theory,  1 26 

Biirker's  hsemocytometer,  228 

Butter,  composition  of,  141,  238 

Butyric  acid,  8,  238,  243,  284,  309 

Butyrin,  141,  238 

Bynin,  93,  112 

Cadaverin,  8  s 

Calcium  and  magnesium  in  urine,  284,  320 
carbonate  in  urinary  sediments,  362,  363 
casein,  128,  236 
oxalate,  362 

in  urinary  sediments,  362 
phosphate  in  urinary  sediments,  362,  364 

in  milk,  235,  242 
sulphate  in  urinary  sediments,  362,  364 
Calculi,  biliary,  161,  165 
urinary,  379 

calcium  carbonate  in,  380 

oxalate  in,  380 
cholesterol  in,  382 
cystine  in,  360 
fibrin  in,  380 
indigo  in,  382 
phosphates  in,  380 
uric  acid  and  urates  in,  380 
urostealiths  in,  380 
xanthine  in,  3-80 
Calliphora,     larvae    of,    formation    of    fat    from 

protein  by,  143 
Cane  sugar  (see  Sucrose,  p.  46) 
Canton  silk,  64,  74 
Caproic  acid,  23s,  238 
Carbamic  acid,  196 
Carbocyclic  nucleus,  73,  74 
Carbohydrates,  25 

classification  of,  25 
composition  of,  25 
review  of,  57 

scheme  for  detection  of,  58 
variation  in  solubility  of,  26 
Carbonates  in  urine,  284,  321 
Carbon  moiety  of  protein  molecule,  142 
Carbon  monoxide,  haumoglobin,  216 

tannin  test  for,  217 
Carboxyl  group,  4,  75,  95 


INDEX. 


459 


Carboxylase,  4,  9,  36,  .5,?i,  389 
Carnine,  255 
Carnitine,  255 

formula  for,  260 
Camomuscarine,  255 
Camosine,  255,  260 
Cartilage,  250 

constituents  of,  250 

experiments  on,  250 

Hopkins-Cole  reaction  on,  251 

loosely  combined  sulphur  in,  251 

Millon's  reactiorPon,  251 

preparation  of  gelatin  from,  251 

solubility  of,  250 

xanthoproteic  test  on,  251 
Casein,  72,  128,  236 

calcium,  i  28.  236 

decomposition  of,  72 

quantitative  determination  of,  438,  439 

soluble,  128,  236 
Caseinogen,  94,  95,  128,  236,  438 

action  of  rennin  upon,  128,  236 

biuret  test  on,  241 

Millon's  test  on,  241 

precipitation  of,  241 

preparation  of,  241 

quantitative  determination  of,  Hart's  method 
for,  439 

solubility  of,  241 

test  for  loosely  combined  sulphur  in,  241 

test  for  phosphorus  in,  242 
Casts,  369.  371 

blood,  369,  3  73 

epithelial,  369,  373 

fatty,  369,  3  73 

granular,  369,  372 

hyaline,  369,  371 

pus,  369,  3  74 

waxy.  369,  373 
Casts  in  urinary  sediments,  369,  371 
Cat  gut,  13s 
Catalase,  4,  17,  23 

experiments  on,  17,  23 

quantitative  determination  of,  23 
Catalysis,  2 
Cellulose,  26,  S3 

action  of  Schweitzer's  reagent  on,  54 

hydrolysis  of,  54 

iodine  test  on,  54 

solubility  of,  54 

solvents,  55 

utilization  by  animals,  53 
Cellulose  group,  26 
Cerebrin  (cerebroside),  268,  272 

experiments  on,  272 

hydrolysis  of,  272 

microscopical  examination  of,  273 

preparation  of,  272 

solubility  of,  272 
Cerebro-spinal  fluid,  choline  in,  269 
Cerebrosides,  268 
Charcot-Leyden  crystals,  18 1 

form  of,  181 
Chlorides  in  urine,  284,  316 
detection  of,  317 

quantitative  determination  of,  417 
Cholecyanin,  16 1 
Choleprasin,  160 


Cholera-red  reaction  for  indole,  176 
Cholesterol,  162,  165,  268.  271,  362,  366 
crystalline  form  of,  1 66 
formula  for,  270 

iodine-sulphuric  acid  test  for,  165,  272 
isolation  of,  from  biliary  calculi,  165 
Liebermann-Burchard  test  for,  165,  272 
occurrence  of,  in  urin^y  sediments,  362,  366 
origin  of,  271 

preparation  of,  from  nervous  tissue,  271 
Salkowski's  test  for,  166,  272 
SchifT's  reaction  for,  166,  272 
tests  for,  i6s,  272 
Choletelin,  160 
Choline,  269,  273 

formula  for,  269 
tests  for,  273 
Chondrigen,  1 13 
Chondroalbumoid,  250 
Chondromucoid,  113,  250 
Chondroitin,  250 

Chondroitin-sulphuric  acid,  250,  283,  303 
Chondrosin,  250 

Chromoproteins,  see  Hemoglobins,  94,  95 
Chyle,  206 

CipoUina's  test,  29,  325 
Clark's     modification     of     Dehn's     method     for 

determination  of  chlorides,  417 
Cleavage    products    of    protein    (see    Decompo- 
sition products),  69,  72 
Clupeine,  72,  93,  95 
Coagulated  proteins,  94,  117 
biuret  test  on,  1 19 
digestion  of,  20 
formation  of,  1 1  7 
Hopkins-Cole  reaction  on,  1 19 
Millon's  reaction  on,  119 
solubility  of,  119 
xanthoproteic  reaction  on ,  119 
Coagulation  of  blood,  203 

Howell's  theory  of,  203 
Coagulation  of  proteins,  106,  117 

changes  in  composition  during,  117 
fractional,  106,  117 
Coagulation  temperature  of  proteins,  106,  117 
apparatus  used  in  determining,  106 
method  employed  in  determining,  106 
Co-enzyme,  7,12 
Collagen,  93,  112,  247 
experiments  on,  247 
percentage  of,  in  ligament,  249 

in  tendon,  246 
production  of  gelatin  from,  248 
solubility  of.  248 
transformation  of,  247 
Collodion  dialyzer,  30 
Colloidal  solution,  23s 
Colloids,  235,  2S5 

tissue,  268 
Colostrum,  236,  238 

microscopical  appearance  of,  239 
Combined  hydrochloric  acid  (protein  salt),  61,  126 
130,  I3S,  445 
preparation  of,  445 
tests  for,  130 
Compound  test  for  lactose  in  urine.  355 
Congealing-point  of  fat,  147 
Congo  red,  as  indicator,  131,  132 


460 


INDEX. 


Congo  red,  preparation  of,  132 
Conjugated  proteins,  94,  95,  112 
classes  of,  94,  95,  112 
nomenclature  of,  94,  112 
occurrence  of,  112 
Conjugate  glycuronates,  32,  323,  327,  351 

fermentation-reduction  test  for,  351 
Tollens'  reaction  on,  352 
Connective  tissue,  245 
Constipation,  aid  in,  56,  180 
Cowie's  guaiac  test,  186 
Creatine,  196,  255,  257,  283,  323,  416 
crystalline  form  of,  254 
formula  for,  260 

quantitative  determination  of,  416 
separation  of,  from  meat  extract,  264 
Creatinine,  32,  255,  283,  294,  415 
coefficient,  definition  of,  294 
crystalline  form  of,  295 
daily  excretion  of,  294 

as   influenced    by    muscular   tonus, 
296 
experiments  on,  296 
formula  for,  260,  294 
Jaffe's  reaction  for,  297 
quantitative  determination  of,  415 
Salkowski's  test  for,  297 
separation  of,  from  urine,  296 
Weyl's  test  for,  296 
Creatinine-zinc  chloride,  formation  of,  295,  296 
Cresol,  para,  169 
tests  for,  177 
Cross  and  Sevan's  reagent,  54 
preparation  of,  54 
solubility  test,  S4 
Cryoscopy,  279 
Cul-de-sac,  125 
Cupri-potassium  biuret,  formation  of,  99 

formula  for,  99 
Cyanuric  acid,  286 

formula  for,  286 
Cylindroids  in  urinary  sediments,  369,  376 
a-Cyprinine,  73 
Cystine,  72,  74,  80,  362,  366 
crystalline  form  of,  81 
detection  of,  366 
formula  for,  80 

in  urinary  sediments,  363,  366 
Cytoglobulin,  94,  95 
Cytosine,  113 

Wheeler- Johnson  reaction  for,  113 

Dakin's  methods  for  quantitative  determination 

of  hippuric  acid,  406 
Dare's  haemoglobinometer,  222 

description  of,  222 

determination  of  haemoglobin  by,  222 
Darmstadter's     method     for     determination     of 

^-oxybutyric  acid,  426 
Deamidizing  enzyme,  3,  4 
Decomposition  products  of  proteins,  68,  69,  72,  76 

crystalline  forms  of,  77-89 

experiments  on,  89 

isolation  of,  89 
Degradation  products  of  protein  (see  Decompo- 
sition products,  68) 
Dehn-Clark  method  for  chlorides,  417 
Dehn's  reaction  for  hippuric  acid,  300 


Delusive  feeding  experiments,  125 

Derived  proteins,  94,  114 

Detection  of  preservatives  in  milk,  243 

boric  acid  and  borates,  244 

formaldehyde,  243 

hydrogen  peroxide,  244 

salicylic  acid  and  salicylates,  243 
Deuteroproteose,  94,  95,  121 
Dextrin,  26,  48,  52 

achroo-,  48,  61 

a-achroo-,  61 

/3-achroo-,  61 

/•-achroo-  61 

erythro-,  48,  61 

action  of  tannic  acid  on,  53 

diffusibility  of,  53 

Fehling's  test  on,  53 

hydrolysis  of,  53 

iodine  test  on,  52 

solubility  of,  52 
Dextrosazone,    crystalline    form    of,    Plate    III, 

opposite  p.  28 
Dextrose,  25,  27,  323 

Allen's   modification    of   Fehling's   test   for, 
329 

Barfoed's  test  on,  36,  331 

Boettger's  test  on,  34,  330 

Bottu's  test  on,  29,  325 

CipoUina's  test  on,  29,  325 

Benedict's    modification    of   Fehling's    test, 
33, 328 

diffusibility  of,  30 

experiments  on,  27,  324 

Fehling's  test  on,  32,  327 

fermentation  of,  35,  331 

formula  for,  27 

iodine  test  on,  29 

Molisch's  reaction  on,  27 

Moore's  test  on,  30 

Nylander's  test  on,  34,  330 

phenylhydrazine  test  on,  28,  324 

quantitative  determination  of,  384 

reduction  tests  on,  30,  325 

Riegler's  reaction,  29,  325 

solubility  of,  27 

Trommer's  test  on,  31,  326 
Dextrosazone,    crystalline    form    of,    Plate    III, 

opposite  p.  28 
Diacetic  acid,  323,  348 

Amold-Lipliawsky  test  for,  349 
formula  for,  348 
Gerhardt's  test  for,  348 
quantitative  determination  of,  425 
Diamino  acid  nitrogen,  69 
Diaminotrihydroxydodecanoic  acid,  72,  73,  89 
a-£-di-amino-caproic  acid,  73,  8s 
Dialysis,  30 

Dialyzers,  preparation  of,  30 
Diastase  (see  Vegetable  amylase,  3,  4,  10) 
Diazo-benzene-sulphonic  acid,  359 

reagent,  preparation  of,  359 
Diazo  reaction  (Ehrlich's),  359 
Differentiation  between  pepsin  and  pepsinogen, 

i27>  134 
Digestion,  gastric,  124 

pancreatic,  148 

salivary,  59 
Di-iodo-hydroxypropane  (lothion),  35,  330 


INDEX. 


461 


Di-methyl-amino-azobenzene    (see    Topfer's    re- 
agent), 13  a 

Dipeptides,  62,  71,  75,  95 

Disaccharides,  25,  43 
classification  of,  25 

Dissociation    products    of    protein    (see    Decom- 
position products,  68) 

Doremus-Hinds  ureometer,  395 

Drying  method  for  determination  of  total  solids 
in  urine,  434 

Duodenum,  epithelial  cells  of,  148 

Earthy  phosphates  in  urine,  284,  317 

quantitative  determination  of,  413 
Edestan,  20,  94,  115 

experiments  on,  115 
Edestin,  72,  93,  109 

coagulation  of,  in 

crystalline  forms  of,  no 

decomposition  of,  72 

microscopical  examination  of,  in 

Millon's  test  on,  in 

preparation  of,  no 

solubility  of,  in 

tests  on  crystallized,  in 
filtrate  of,  in 
Ehrlich's  diazo-benzene-sulphonic   acid   reagent, 

preparation  of,  359 
Ehrlich's  diazo  reaction,  359 
Ehrlich's  mechanical  eye-piece,  use  of,  228 
Einhom's  bead  test,  189 
Einhom's  saccharometer,  36 
Elastin,  93,  112,  249 

adsorption  of  pepsin,  by,  249 

experiments  on,  249 

preparation  of,  249 

solubility  of,  249 
Electrical  conductivity  of  urine,  281 
Electrolj-tes,  influence  on  enzyme  activity,  7,  151 
Embryos,  glycogen  in,  256 
Emulsin,  4 
Enterokinase,  4,  150 
Enzymes,  i 

activation  of,  6 

adsorption  of,  6 

classification  of,  4 

definition  of,  2 

experiments  on,  10 

influence  of  electrolj-tes,  7,  151 

list  of,  4 

preparation  of,  5 

properties  of,  5 

reference  books,  9 
Epiguanine,  284,  312 
Episarkine,  284,  312 
Epithelial  cells  in  urinary  sediments,  369 

casts  in  urinary  sediments,  369,  370 
Epithelial  tissue,  24s 

experiments  on,  245 
Erepsin,  4,  15,  152 

experiments  on,  15 
Erythrocytes,  194,  196,  197,  224 

counting  the,  224,  228 

diameter  of,  196 

form  of,  196 

influence  of  osmotic  pressure  on,  208 

in  urinary  sediments,  369,  376 

number  of,  per  cubic  mm.,  197 


Erythrocytes  of  diflerent  species,  196 

stroma  of,  201 

variation  in  number  of,  197 
Erythro-dextrin,  48,  6 r 
Esbach's  albuminometer,  384 

method  for  determination  of  albumin,  384 

reagent,  preparation  of,  384 
Ester,  definition  of,  139 

hydrochloric  acid,  of  ha;matin,  212 

sulphuric  acid,  of  haematin,  212 
Ethereal  sulphates,  283,  297 

quantitative  determination  of,  409 
Ethereal  sulphuric  acid,  169,  283,  297 
Ethyl  butyrate  test  for  pancreatic  lipase,  157 
Euglobulin,  194 
Excelsin,  no 

crystalline  form  of,  no 
Extractives  of  muscular  tissue,  255 
nitrogenous,  255 
non-nitrogenous,  255 

Fatigue  substances  of  muscle,  260 
Fats,  139 

absorption  of,  141 

apparatus    for    determination    of    melting- 
point  of,  146 

chemical  composition  of,  139,  140 

congealing-point  of,  146 

crystallization  of,  141,  144 

digestion  of,  141 

emulsification  of,  141,  143 

experiments  on,  143 

formation  of  from  protein,  142 

formation  of  acrolein  from,  143 

hydrolysis  of,  140 

in  milk,  23s,  238,  242 

in  urine,  323,  353,  369,  373 

melting-point  of,  146 

nomenclature  of,  140 

occurrence  of,  139 

permanent  emulsions  of,  141,  143 

quantitative  determination  of,  in  milk,  435 

rancid,  141 

reaction  of,  141 

saponification  of,  140,  144 

solubiUty  of,  141,  143 

transitory  emulsions  of,  141,  143 
Fat-splitting  enzymes  (see  Lipases,  3,  4,  12) 
Fatty  acid,  139,  140,  145 
Fatty  casts  in  urinary  sediments,  369,  373 
Fatty  degeneration,  142 

Fecal  amylase,  quantitative  determination  of,  189 
Fecal  bacteria,  181,  191 

Fecal  bacteria,  quantitative  determination  of,  191 
Feces,  178 

agar-agar,  influence  of,  180 

bacteria  in,  181,  191 

blood  in,  181,  185 

daily  excretion  of,  178 

detection  of  albumin  and  globulin  in,  188 
bile  acids  in,  187 
bilirubin  in,  187 
caseinogen  in,  187 
cholesterol  in,  185 
hydrobilirubin  in,  186 
inorganic  constituents  of,  188 
nucleoprotein  in,  187 
proteose  and  peptone  in,  188 


462 


INDEX. 


Feres,  enzymes  of,  182 

experiments  on,  184 

"fasting,"  183 

form  and  consistency  of,  180 

hydrogen  ion  concentration  of,  180 

macroscopic  constituents  of,  181 

microscopic  constituents  of,  181 

nitrogen  of,  182 

odor  of,  179 

parasites  and  ova  in,  183 

pigment  of,  178 

reaction  of,  1 80 

Scybala  form  of,  180 

separation  of,  importance  of,  180,  189 

separation  of,  experiment  on,  189 
Fehling's  method  for  determination  of  dextrose, 

384 
Benedict's     modifications     of, 

38s 
solution,  preparation  of,  32,  327 
test,  32,  327 

Allen's  modification  of,  329 
Benedict's  modifications  of,  3  28 
Ferments,  classification  of,  4 
Fermentation,  "  sugar-free,"  9,  36 
Fermentation   •  method     for     determination     of 

dextrose,  388 
Fermentation-reduction       test       for       conjugate 

glycuronates,  351 
Fermentation  test,  36,  331 

Ferric  chloride  test  for  thiocyanate  in  saliva,  64 
Fibrin,  19s,  203,  214,  369,  378 

in  urinary  sediments,  369,  378 
separation  of,  from  blood,  195,  203 
solubility  of,  214 
Fibrin  ferment,  19s,  203 
Fibrin-heteroproteose,  73 
Fibrinogen,  19s,  203 
Fibroin,  Tussah  silk,  73 
Fischer  apparatus,  80 

photograph  of,  80 
Fleischl's       hsemometer,  220 
description  of,  220 

determination  of  ha;moglobin  by,  220 
Pleischl-Miescher  hsemometer,  221 
Fluorides  in  urine,  284,  322 
Fly-maggots,  experiments  on,  142 
Folin-Hart    method    for   determination    of   com- 
bined acetone  and  diacetic  acid,  421 
for  determination  of  diacetic  acid,  425 
Folin-Messinger-Huppert      method      for      deter- 
mination of  diacetic  acid,  425 
Folin's  method  for  determination  of  acetone,  423 
acidity  of  urine,  427 
ammonia,  399 
creatinine,  415 
ethereal  sulphates,  409 
inorganic  sulphates,  409 
total  sulphates,  408 
urea,  394 
Folin  and  Denis'  method  for  urea,  398 
Folin  and  Macallum's  method  for  ammonia,  400 
Folin  and  Pettibone's  method  for  urea  (No.   i), 

397 
Folin  and  Pettibone's  method  for  urea  (No.  2), 

397 
Folin,    Benedict  and   Myers'   method   for  deter- 
mination of  creatine,  416 


Folin-Shaffer  method  for  determination  of  uric 

acid,  389 
Foreign  substances  in  urinary  sediment,  369,  3  78 
Formation  of  methylphenylkevulosazone,  40 
Form  elements  of  blood,  194 
Formic  acid,  284,  309 
Fractional  coagulation  of  proteins,  117 
Free  hydrochloric  acid,  126 
tests  for,  130 
Freezing-point  of  bile,  159 

blood,  194 

milk,  23  s 

pancreatic  juice,  149 

urine,  279 
Fuchsin-frog  experiment,  262 
Fuld  and  Levison's  method  for  peptic  activity, 

20 
Fundus  glands,  125 
Furfurol  solution,  preparation  of,  164 
Fusion  mixture,  preparation  of,  271 

Galactans,  26,  55 

Galactase,  239 

Galactose,  25,  41,  323,  355 

experiments  on,  41 
Gallic  acid  test  for  formaldehyde,  243 
Ganassini's  test,  293 
Gastric  digestion,  124 

conditions  essential  for,  134 

general  experiments  on,  134 

influence  of  bile  on,  136 

influence  of  water  on,  124 

influence  of  different  temperatures  on, 

134 
most  favorable  acidity  for,  134 
power  of  different  acids  in,  13s 
products  of,  127,  130 
Gastric  fistula,  125 
Gastric  juice,  125 

acidity  of,  126 

influence  of  water  on,  124 
artificial,  preparation  of,  129 
composition  of,  125 
enzymes  of,  125 
lactic  acid  in,  test  for,  136 
origin  of  hydrochloric  acid  of,  126 
quantitative  analysis  of,  440 
quantity  of,  125 
reaction  of,  126 
secretion  of,  124 

influence  of  water  on,  124 
specific  gravity  of,  125 
Gastric  lipase,  129 
Gastric  protease,  1 1 
Gastric  rennin,  125,  128,  136 

action  of,  upon  caseinogen,  128,  136,  236, 

241 
experiments  on,  136,  241 
influence  of,  upon  milk,  136,  241 
in  gastric  juice,  absence  of,  129 
nature  of  action  of,  128,  236 
occurrence  of,  128,  236 
Gelatin,  72,  74,  247,  248 
coagulation  of,  248 
decomposition  of,  72 
experiments  on,  248 
formation  of,  247 
Hopkins-Cole  reaction  on,  248 


INDKX. 


463 


Gelatin,  Millon's  reaction  on,  J48 

precipitation  of,  by  alcohol,  249 
alkaloidal  reagents,  248 
metallic  salts,  248 

precipitation  of,  by  mineral  acids,  248 

preparation  of,  from  cartilage,  251 
from  collagen,  248 

salting-out  of,  248 

solubility  of,  248 
Gerhardt's  test  for  diacetic  acid,  348 
Gerhardt's  test  for  urobilin,  311 
Gies'  biuret  reagent,  preparation  of,  99 
Gliadin,  93,  112 

decomposition  of,  72 
Globin,  72,  93,  197 

decomposition  of,  72 
Globulins,  93,  95,  109 

experiments  on,  109 

preparation  of,  109 

serum,  93.  194.  3^i.  332 
in  urine,  323,  332 
tests  for,  332 

vegetable,  109 
Glucoproteins  (see  Glycoproteins,  p.  94,  112) 
Glucose  (see  Dextrose,  p.  25,  27,  323) 
r.lucothionic  acid,  371 
Glutamic  acid,  74,  87,  149 

formula  for,  87 
Glutelins,  93,  95,  1 1 1 
Glutenin,  93,  95,  m 
Glycerol,  139,  146 

borax  fusion  test  on,  146 

experiments  on,  146 

formula  for,  143 
Glycerol   extract  of  pig's  stomach,   preparation 

of,  130 
(jlycerophosphoric  acid,  268,  269,  284,  310 
Glycocholic  acid,  159 
Glycocholic  acid  group,  159 
Glycocoll,  74,  77,  159,  168 

crystalline  form  of,  167 

formula  for,  77,  159,  168 

preparation  of,  167 
Glycocoll    ester   hydrochloride,    crystalline  form 

of,  7  7 
Glycogen,  26,  52,  255,  256 

experiments  on,  263 

hydrolysis  of,  264 

in  embryos.  256 

influence  of  saliva  on,  264 

iodine  test  on,  263 

preparation  of,  263 
Glycogenase,  4 
Glycol>'tic  enzymes,  4 
Glycoproteins,  94,  112,  247 

experiments  on,  247 

hydrolysis  of,  247 
Glycosuria,  alimentary,  27 
Glycosuric  acid,  306 

(jlycuronates,  conjugate,  32,  323,  327,  351 
Glycuronic  acid,  42,  353 
Glycyl-glycine,  formation  of,  75 
Glycyl-tryptophane  test,  15,  154 
Glyoxylic  acid,  97,  98 

formula  for,  97,  98 
Gmelin's  test  for  bile  pigments,  162,  342 

Rosenbach's  modification  of,  162,  342 
Granular  casts  in  urinary  sediments,  369,  372 


Granulose,  48 

Green  stools,  cause  of,  179 

Gross'  method  for  quantitative  determination  of 

tryptic  activity,  22 
Guaiac  solution,  preparation  of,  446 
Guaiac  test  on  blood,  204,  209 

on  feces,  186 
milk,  240 
pus,  371 

in  urine,  341 
Guaiac  test,  Schumm's  modification  of,  209 
Guanase,  4 

Guanidine-a-amino- valerianic  acid,  73,  83 
Guanidine-residue,  69 
Guanine,  4,  255,  261 
Gum  arabic,  26,  55 

Gums  and  vegetable  mucilage    group  of   carbo- 
hydrates, 26 
Gunning's  iodoform  test  for  acetone,  346 
Giinzberg's  reagent,  as  indicator,  131 

preparation  of,  131 
Gurber's  reaction  for  indican,  299 

Haemagglutination,  197,  208 
Haemagglutinin,  197,  208 
Haematin,  114,  197,  212 

^cid-,  219 

alkali-,  218 

preparation  of,  212 

reduced  alkali-,  218 
Haematoidin,  160,  161,  179 

crystalline  form  of,  161,  179 

in  urinary  sediments,  362,  367 
Hsematuria,  339 
Haematoporphyrin,  202,  219,  323,  353 

in  urine,  274,  323,  353 
Hsemin  crystals,  form  of,  204 

test,  210 
Haemochromogen,  114,  197,  213 
Haemocyanin,  94,  95,  114 
Hjemocytometer,  Biirker's,  228 

Thoma-Zeiss,  224 
Hasmoconein  (see  Blood  dust,  194,  203) 
Haemoglobin,  94,  95,  112,  114 

carbon  monoxide,  202,  216 

decomposition  of,  197 

diffusion  of,  209 

met,  202,  2t8 

oxy,  197,  201,  216 

quantitative  determination  of,  220,  222 

reduced.  197,  216 
Haemoglobins,  94,  112 
Haemoglobinuria,  316 
Haemolysis,  194,  207 
Hffiser's  coefficient,  279,  434 
Hair,  human,  74,  245 
Hammerschlag's    method    for   determination    of 

specific  gravity  of  blood,  206 
Hammarsten's  reaction,  163,  343 

reagent,  preparation  of,  163,  343 
Hart's  caseinogen  method,  439 
Hayem's  solution,  229 
Hay's  test  for  bile  acids,  164,  344 
Heintz  method  for  determination  of  uric  acid, 

290,  390  1^ 

Helicoprotein,  94 

Heller's  test  for  blood  in  urine,  340 
Heller-Teichmann  reaction  for  blood  in  urine,  340 


464 


INDEX. 


Heller's  ring  test  for  protein,  104,  333 
Hemicellulose,  26,  ss 
experiments  on,  56 
utilization  of,  by  animals,  55 
Hemiurate,  365 

Herter's  naphthaquinone  reaction  for  indole,  175 
Herter's  para-dimethylaminobenzaldehyde  reac- 
tion, 176 
Heterocyclic  nucleus,  73,  74 
Heteroproteose,  95 
Heteroxanthine,  284,  313 
Hexone  bases,  8s 
Hexosans,  26,  55 
Hexoses,  25,  26 

Hippuric  acid,  168,  283,  300,  406 
crystalline  form  of,  300 

Dakin's    method    for    quantitative    determi- 
nation of,  406 
Dehn's  reaction  for,  302 
experiments  on,  168,  277,  301 
formula  for,  168,  300 
in  urinary  sediments,  367 
melting-point  of,  302 
Roaf's  method  for  crystallization  of,  301 
separation  of,  from  urine,  301 
solubility  of,  302 
sublimation  of,  302 
synthesis  of,  168 
Histidine,  73,  82,  149 

hydrochloride,  crystalline  form  of,  83 
Knoop's  color  reaction  for,  82 
Histones,  93,  95 

Hoffmann's  reaction  for  tyrosine,  91 
Homogentisic  acid,  32,  283,  306,  327 

formula  for,  306 
Hopkins-Cole  reaction,  98 

on  solutions,  98 
on  solids,  108 
Hopkins-Cole  reagent,  preparation  of,  98 
Hopkins-Cole   reagent    (Benedict    modification), 

preparation  of,  98 
Hordein,  73,  93,  112 
Horismascope  (see  Albumoscope,  104) 
Hormones,  definition  and  discussion  of,  148,  237 
Hopkins'  thiophene  reaction  for  lactic  acid,  137 
Hiifner's  urea  apparatus,  394 
Human  fat,  composition  of,  140 

hair,  composition  of,  245 
Huppert's  reaction  for  bile  pigments,  162,  342 
Hurthle's  experiment,  267 
Hyaline  casts  in  urinary  sediments,  369,  371 
Hydrobilirubin,  detection  of,  in  feces,  179 

extraction  of,  186 
Hydrochloric  acid  of  the  gastric  juice,  126 
origin  of,  theories  as  to,  1 26 
seat  of  formation  of,  125 
Hydrochloric  acid  test  for  formaldehyde  (Leach) , 

243 
Hydrogen  peroxide  in  urine,  284,  322 

detection  of,  in  milk,  244 
Hydrolysis  of  cellulose,  54 
cerebrin,  273 
dextrin,  53 
glycogen,  264 
inulin,  51 
proteins,  69 
starch,  50 
sucrose,  47 


Hyperacidity,  126 

Hypoacidity,  126 

Hypobromite  solution,  preparation  of,  392 

Hypoxanthine,  255,  261,  265,  284,  312 

formula  for,  261 
Hypoxanthine  silver  nitrate,  crystalline  form  of, 
26s 

Ichthulin,  94 
Ignotine,  255 

formula  for,  260 
Imide  bonds,  75 
Iminazolethylamine,  171 
Iminazolpropionic  acid,  171 
Indican,  169,  283,  297,  416 

Barberio's  reaction,  300 

formula  for,  169,  298 

Gurber's  reaction  for,  299 

Jaffe's  test  for,  298 

Lavelle's  reaction  for,  299 

Obermayer's  test  for,  299 

origin  of,  169,  297  • 

Rossi's  reaction  for,  299 
Indicators,  theory  of,  130 

table  of,  131 
Indigo-blue,  170,  299 

formula  for,  170,  299 
Indigo  in  urinary  sediments,  362,  368 
Indolacetic  acid,  171,  407 
Indole,  169,  17s,  179 

formula  for,  169 

origin  of,  169,  179 

test  for,  17s 
Indole- a-amino-propionic  acid,  73,  82 
Indolpropionic  acid,  171 
Indoxyl,  169,  299 

formula  for,  169,  299 

origin  of,  169,  298 

potassium  sulphate    (see   Indican,    pp.    169, 
283,  297,  416 
Indoxyl-sulphuric  acid,  169,  298 

formula  for,  169,  298 
Infraproteins  (see  Metaproteins,  94,  95,  ns) 
Inorganic  physiological  constituents  of  urine,  284, 

313 
Inosinic  acid,  255,  260 

formula  for,  260 
Inosite,  25,  323,  357 

formula  for,  357 

in  urine,  323,  357 
Intestinal  juice,  150 

enzymes  of,  152 

preparation  of,  13 
Inulase,  51 
Inulin,  26,  so 

action  of  amylolytic  enzymes  on.  Si.  65 

Fehling's  test  on,  51 

hydrolysis  of,  si 

iodine  test  on,  51 

reducing  power  of,  51 

solubility  of,  51 

sources  of,  50 
Inversion,  46,  48 
Invertase  (see  Sucrase,  s.  46) 
Invertases,  experiments  on,  13 
Invertin  (see  Sucrase,  4,  5,  46) 
Inverting  enzymes,  3 
Invert  sugar,  46 


INDKX. 


405 


Iodide  of  dextrin,  s» 

of  starch,  50 
Iodine  test,  29,  50,  S«i  S3.  54.  56 
Iodine-sulphuric  acid  test  for  cholesterol,  165,  272 
lodine-zinc-chloride  reaction,  54 
Iodoform  test  for  alcohol,  47 
lodothymol  compound,  347 
"lothion,"  35,  330 
Iron  in  blood,  207 

detection  of,  207 

in  bone  ash,  253 

detection  of,  253     "* 

in  protein,  68 

in  urine,  284,  321 

detection  of,  321 
Isoleucine,  73,  85 
Isomaltose,  25,  44,  61 
Iso valerianic  acid,  171 

Jacoby-Solms  method,  21 

Jaffe's  reaction  for  creatinine,  297 

Jaflfe's  test  for  indican,  298 

V.  Jaksch-Pollak  reaction  for  melanin,  358 

Jejunum,  epithelial  cells  of,   148 

Jolles'  reaction  for  protein,  105,  334 

reagent,  preparation  of,  105,  334 
Juice,  gastric,  124,  125,  127 

pancreatic,  148,  150 

intestinal,  150 

Kantor  and  Gies's  biuret  paper,  99 
Kastle's  peroxidase  reaction,  240 
Kephalin,  268,  270 
Kephyr,  45 

Keratin,  93,  112,  245,  246 
experiments  on,  246 
solubility  of,  246 
sources  of,  245 
sulphur  content  of,  245,  246 
Ketone,  25,  30 
Ketose,  25 
Kjeldahl  method  for  determination  of  nitrogen, 

401 
Kjeldahl-Folin-Farmer  nitrogen  method,  402 
Knoop's  color  reaction  for  histidine,  82 
Knop-Htifner  hypobromite  method  for  determi- 
nation of  urea,  392,  393 
Konto's  reaction  for  indole,  176,  i88 
Koppe's  electrolytic  dissociation  theory.  1  26 
Koumyss,  45 

Kraut's  reagent,  preparation  of,  273 
Kreosotal,  352 

Kruger  and  Schmidt's  method  for  the  quantita- 
tive    determination     of 
purine  bases,  429 
of  uric  acid,.39i 
Kiilz's  test  for  )9-oxybutyric  acid,  351 
Kwilecki's  modification  of  Esbach's  method.  384 
Kynurenic  acid,  283,  307 
formula  for,  307 
isolation  of,  from  urine.  307 
quantitative  determination  of,  307 

Laccase,  4 

Lactalbumin,  93,  235,  238 

quantitative  determination  of,  439 
Lactase,  4,  14,  152 

experiments  on,  14 

30 


Lactic  acid,  45,  136,  256 

ferric  chloride  test  for,  137 
Hopkins'  thiophene  reaction  for,  137 
in  muscular  tissue,  256 
in  stomach  contents,  136,  137 
tests  for,  136 
Uffelmann's  test  for,  136 
Lacto-globulin,  235,  238 
Lactometer,  determination  of  specific  gravity  of 

milk  by,  435 
Lactosazone,  crystalline  form  of,  Plate  III,  oppo- 
site p.  28 
Lactoscope,  Feser's,  438 
Lactose,  25,  45,  23s.  237 

experiments  on,  45 

fermentation  of,  45 

in  urine,  323,  354 

quantitative  determination  of,  440 
Lactosin  in  milk,  238 
Laevo- a-proline,  88 

Laevulosazone,  crystalline  form  of,  Plate  III,  op- 
posite p.  28 
Ljevulose,  25,  39 

Borchardt's  reaction  for,  40 

in  urine,  323,  355 

methyl-phenylhydrazine  test  for,  40 

Seliwanoflf's  reaction  for,  40 
Laiose  in  urine,  323,  358 
"Laked"  blood,  194,  ao? 
Laky  blood,  207 
Laurie  acid,  235 
Laurin,  139 

Lavelle's  reaction  for  indican,  299 
Leach's  hydrochloric  acid  test  for  formaldehyde, 

243 
Lecithans,  94 
Lecithin,  94,  196,  268,  269 

acrolein  test  on,  271 

decomposition  of,  269 

experiments  on,  271 

formula  for,  269 

microscopical  examination  of,  271 

osmic  acid  test  on,  271 

preparation  of,  271 

test  for  phosphorus  in,  271 
Lecithoproteins,  94,  112 
Legal's  reaction  for  indole,  176 

test  for  acetone,  346 
Leucine,  72,  74,  84,  91,  127,  149.  362,  367 

crystalline  form  of  impure,  367 
pure,  8s 

experiments  on,  91 

formula  for,  84 

in  urinary  sediments,  362,  367 

microscopical  examination  of,  91 

separation  of,  from  tyrosine,  90 

solubility  of,  91 

sublimation  of,  91 
Leucocytes,  194,  202 

counting  the,  227 

number  of,  per  cubic  mm.,  202 

size  of,  302 

variation  in  number  of,  202 
Leucocytosis,  202 
Leucosin,  103 

Leucyl-alanyl-glycine,  formation  of,  75 
Leucyl-glycyl-alanine,  62 
Leucyl-leucine,  formation  of,  75 


466 


INDEX. 


Lichenin,  26,  52 

Lieben's  test  for  acetone,  347 

Lieberkuhn's  jelly    (see    Alkali    metaprotein,    p. 

117) 
Liebermann-Burchard   test   for  cholesterol,    165, 

272 
Liebermann's  reaction,  100 
Lipase,  gastric,  125 
Lipase,  pancreatic,  4,  12,  149,  151 

action  of,  in  dilution,  151 

experiments  on,  12,  157 

ethyl-butyrate  test  for,  157 

litmus-milk  test  for,  157 
Lipases,  4,12 

experiments  on,  12 
Lipeses,  3 
Lipins,  268 

Lipoids  of  nervous  tissue,  268,  271 
Lipolytic  enzymes  (see  Lipases,  p.  4,  12). 
"Litmus-milk"  test  for  pancreatic  lipase,  157 
Long's  coefficient,  278,  434 
Lugol's  solution,  preparation  of,  10 1 
Lymph,  194,  205 
Lysine,  72,  73,  85,  127,  149 
Lysine  picrate,  crystalline  form  of,  86 

Magnesia  mixture,  preparation  of,  313,  414,  448 
Magnesium  in  urine,  284,  320 

phosphate  in  urinary  sediments,  362,  368 
Maltase,  4,  15,  44,  62,  152 

experiments  on,  15 
Maltosazone,  crystalline  form  of,   Plate   III,  op- 
posite p.  28 
Maltose,  25,  44 

experiments  on,  44 
structure  of,  44 
Marshall's  urea  apparatus,  392 
Melanin  in  urine,  323,  358 

urinary  sediments.  362,  368 
Melting-point  apparatus,  146 

of  fats,  determination  of,  146 
Messinger-Huppert  method   for  determination   of 

combined  acetone  and  diacetic  acid,  422 
Metaproteins,  94,  115 
acid,  1 16 
alkali,  1 17 
experiments  on,  116 
precipitation  of,  116 
sulphur  content  of,  116 
Methaemoglobin,  202,  218 
Methylene  blue,  135 
Methyl-mercaptan,  169 
Methyl-pentose  (see  Rhamnose,  p.  25) 
Methylphenylhydrazine.  40 
Methylphenyllaivulosazone,  formation  of,  40 
i-methylxanthin,  284,  312 
Mett's     method     for    determination     of     peptic 

activity,  19 
Mett's  tubes,  preparation  of,  20 
Micro-organisms  in  urinary  sediments,  369,  378 
Milk,  23s 

citric  acid  in,  235 

detection  of  calcium  phosphate  in,  242 
lactose  in,  242 
preservatives  in,  243 
difiference  between  human  and  cow's,  237 
experiments  on,  239 
formation  of  film  on,  236,  239 


Milk,  freezing-point  of,  235 
guaiac  test  on,  240 
influence  of  rennin  on,  241 
isolation  of  fat  from,  242 
Kastle's  peroxidase  reaction  on,  240 
Lactose  in,  235,  237,  242 

crystalline  form  of,  238 
microscopical  appearance  of,  236,  239 
preparation  of  caseinogen  from,  241 
properties  of  caseinogen  of,  241 
quantitative  analysis  of,  43  s 
reaction  of,  23s,  239 

separation  of  coagulable  proteins  of,  242 
specific  gravity  of,  235,  239 
Millon's  reaction,  97 

reagent,  preparation  of,  97 
Mohr's   method   for   determination    of   chlorides, 

418 
Molisch's  reaction,  27 
Molybdic  solution,  preparation  of,  64 
Monamino  acid  nitrogen,  69 
Monosaccharides,  25,  26 

Barfoed's  test  for,  36,  331 
classification  of,  25 
Moreigne's  reaction  for  uric  acid,  293 

reagent,  preparation  of,  293 
Morner-Sjoqvist-Folin  method  for  determination 

of  urea,  395 
Morner's  reagent,  preparation  of,  91 

test  for  tyrosine,  91 
Motor  and  functional  activities  of  the  stomach, 

136 
Mucic  acid,  41,  45,  354,  355 

test,  41,  45,  354,  355 
Mucin,  60,  63,  94,  112 
biuret  test  on,  63 
hydrolysis  of,  64 
isolation  of,  from  saliva,  63 
Millon's  reaction  on,  63 
Mucins,  94,  112 
Mucoid,  94,  112,  246,  247,  249 
experiments  on,  247 
hydrolysis  of,  247 
in  urine,  308,  323,  319 
preparation  of,  from  tendon,  247 
Mucoids,  94,  1 12 
Murexide  test,  292 
Muscle  plasma,  254,  262 

formation  of  myosin  clot  in,  254,  262 
fractional  coagulation  of,  254,  261 
preparation  of,  262 
reaction  of,  254,  261 
Muscular  tissue,  254 

ash  of,  smooth  and  striated,  259 
commercial  extracts  of,  260 
experiments  on  "dead,"  263 

"living,"  261 
extractives  of,  255,  264 
fatigue  substances  of,  260 
formulas  of  nitrogenous  extractives  of, 

260 
glycogen  in,  256,  263 
involuntary,  254 
lactic  acid  in,  255,  256 
nonstriated,  254 
pigment  of,  260 

preparation  of  glycogen  from,  263 
muscle  plasma  from,  261,  262 


INDEX. 


467 


Muscular  tissue,  proteins  of,  254 
reaction  of  living,  257 
rigor  mortis  of,  254 
separation  of  extractives  from,  264 
striated,  254 
voluntary,  254 
Myohaematin,  260 
Myosan,  94 

formation  of,  26,? 
Myosin,  254 

biuret  test  gn,  26,? 
coagulation  of,  26,5 
preparation  of,  26.5 
solubility  of,  26.? 
Myosinogen,  254 
Myristic  acid,  235 
Myristin,  139 
Myrtle  wax  (see  Bayberry  tallow.  144) 

Nakayama's  reaction  for  bile  pigments,  162.  342 

reagent,  preparation  of,   162,  342 
Nencki  and  Sieber's  reaction  for  urorosein,  359 
Neosine,  255 

formula  for,  260 
Nervous  tissue.  268 

constituents  of,  268 
experiments  on  lipoids  of,  271 
lipoids  of,  268,  27  I 
percentage  of  water  in,  268 
phosphorized  fats  of,  268 
proteins  of,  268 
Nessler-Winkler  solution,  404 
Neurokeratin,  268 
Neutral  fats,  139,  141,  143 
Neutral  olive  oil,  preparation  of,  143 
Neutral  sulphur  compounds,  283,  303 
Nitrates  in  urine,  284,  322 
Nitrilase,3 
Nitrilese,  3 

Nitrites  in  saliva,  test  for,  64 
Nitrogen,  68 

forms  of  in  protein  molecule,  69 
importance  of,  in  sustaining  life,  69 
in  urine,  quantitative  determination  of,  401 
Nitrogen  distribution,  calculation  of,  402 
Nitrogen  iodide,  formation  of,  346 
Nitrogenous  extractives  of  muscular  tissue,   255, 
264 
formulas  for,  260 
Nitroso-indole  nitrate  test,  i  76 
Nitrosothymol,  formation  of  in  Heller's  test,  ^i^:^ 
Non-nitrogenous  extractives  of  muscular  tissue, 

355 
Normal  urine,  274 

characteristics  of,  274 
constituents  of,  283 
experiments  on,  287 
Novaine.  255 

formula  for,  260 
Nubecula,  308,  339 
Nuclease,  4 
Nucleic  acid,  94,  113 
Nucleins,  113,  130 
Nucleohistone,  93,  95 

Nucleoproteins.  94,  95,    112,   162,   283,  308.  323, 
339 
in  bile,  162 
in  feces,  187 


Nucleoproteins  in  nervous  tissue,  268 

in  urine,  283,  308,  323,  339 
lest  for,  339 

occurrence  of,  i  1 3 

Ott's  precipitation  test  for,  339 
Xylander's  reagent,  preparation  of,  34,  330 

test,  34,  330 

Obermayer's  test  for  indican,  299 

reagent,  preparation  of,  299 
Oblitine,  255 
"Occult"  blood  in  feces,  181,  185 

tests  for,  185 
Olein,  140,  235 
Olive  oil,  143 

emulsification  of.  143 
neutral,  preparation  of,  143 
Opalisjn  in  milk,  238 
Optical  methods,  39,  194 
Orcinol  test.  43,  353 

Organic  physiological  constituents  of  urine,  283 
Organized  ferments,  i 
Organized  urinary  sediments,  361,  369 
Ornithine,  70,  171 
Osborne-Folin  method  for  determination  of  total 

sulphur  in  urine,  410 
Ossein,  25  i 

preparation  of,  251 
Osseoalbumoid,  251 
Osseomucoid,  94,  113,  251 

chemical  composition  of,  113 
Osseous  tissue,  251 

experiment  on,  252 
Ott's  precipitation  test  for  detection  of  nucleo- 

protein  in  urine,  339 
Ovalbumin,  93 
Ovoglobulin,  93 

Oxalated  plasma,  preparation  of,  214 
Oxalic  acid.  283,  302.  433 
formula  for,  302 
in  urine,  283,  302 
quantitative  determination  of,  433 
Oxaluria,  303 
Oxaluric  acid,  283,  308 
Oxamide,  98 
Oxidases,  4,  239 
Oxyacids,  169,  177,  283,  306 

tests  for,  177 
;J-oxybutyric  acid,  323,  349,  42s 

Black's    method    for   determination    of, 

426 
Black's  reaction  for,  350 
formula  for,  349 
Kiilz's  test  for,  351 
origin  of,  350 

polariscopic  examination  for.  351 
quantitative  determination  of.  425 
Shaflfer's  method  for  determination  of, 
425 
Oxyha;moglobin,  69 

Reichert's  method  for  crystallization  of,  214 
crystalline  forms  of,  198-201 
Oxymandelic  acid,  283,  306 
Oxyproline,  72,  73,  88 
Oxyproteic  acid,  283,  303,  359 

Paduschka-Underhill-Kleiner  method  for  quanti- 
tative determination  of  allantoin.  432 


468 


INDEX. 


Palmitic  acid,  140,  145 

crystalline  form  of,  145 

experiments  on,  14s 
formula  for,  140,  151 
preparation  of,  145 
Palmitin,  140,  23s 
Pancreatic  amylase,  4,  150,  155 

digestion  of  dry  starch  by,  151,  is6 
inulin  by,  157 

experiments  on,  155 

influence  of  bile  upon  action  of,  136 
metallic  salts  upon  action  of,  156 

most  favorable  temperature  for  action 
of,  iSS 
Pancreatic  digestion,  148 

general  experiments  on,  154 

products  of,  149,  153 
Pancreatic  insufficiency,  Schmidt's  nuclei  test  for, 

188 
Pancreatic  juice,  140,  148,  149,  150 

artilcial,  preparation  of,  iS3 

daily  excretion  of,  149 

enzymes  of,  149 

freezing-point  of,  149 

mechanism  of,  secretion  of,  148 

reaction  of,  148 

solid  content  of,  149 

specific  gravity  of,  149 
Pancreatic  lipase,  4,  140,  150,  157 

experiments  on,  12,  157 

ethyl-butyrate  test  for,  i  s  7 

litmus-milk  test  for,  i  s  7 
Pancreatic  protease  (see  Trypsin,  p.  11). 
Pancreatic  rennin,  s,  149,  152 

experiments  on,  157 
Papain,  s,  11 
Paracasein,  237 

Para-cresol-sulphuric  acid,  283,  297 
Paradimethylamino  benzaldehyde  solution,  prep- 
aration of,  176 
Paralactic  acid,  236,  284,  309 
Paramyosinogen,  2S4 
Paranucleoprotagon,  268,  271 

Paraoxyphenylacetic  acid,  169,  171,  175,  283,  303 
Paraoxy- ^-phenyl- o-amino-propionic  acid,  74,  79 
Paraoxyphenylpropionic  acid,  169,  171,  i7S.  283, 

303 
Paraphenylenediamine  hydrochloride,  244 
Parasites,  181,  183,  369,  378 
Paraxanthine,  284,  312 
Parietal  cells,  125 
Parotid  glands,  characteristics  of  saliva  secreted 

by,  59 
Pathological  constituents  of  urine,  323 
Pathological  urine,  274,  323 
constituents  of,  323 
experiments  on,  324 
Pektoscope,  379 
Pentamethylenediamine,  1 7 1 
Pentapeptides,  71,  9S 
Pentos3S,  25,  41 

experiments  on,  42 
in  urine,  323,  352 
tests  for,  352 
Pentosans,  26,  41,  55 

Pepsin  (see  Gastric  Protease),  i,  s.  n,  12S.  127 
action  of,  influence  of  bile  upon,  127,  136 
influence  of  different  acids  upon,  135 


Pepsin,  action  of,  influence  of  metallic  salts  upon, 
135 
temperature  upon,  134 
conditions  essential  for  action  of,  134 
differentiation  of,  from  pepsinogen,  127,  134 
digestive  properties  of,  127 
formation  of,  127 

most  favorable  acidity  for  action  of,  134 
presence  of,  in  intestine,  128 
proteolytic  action  of,  127 
Pepsin-hydrochloric  acid,  134 
Pepsin -rennin  controversy,  129 
Pepsinogen,  6,  127,  130 

differentiation  of,  from  pepsin,  127,  134 
formation  of,  127 
extract  of,  preparation  of,  130 
Peptic  activity,  Fuld  and  Levison's  method  for 
determination  of,  20 
Mett's  method  for  the  determination  of, 

19 
Rose's  method  for  determination  of,  21 
Peptic  proteolysis,  127 

products  of,  127 

relation  of,  to  tryptic  proteolysis,  128 
Peptides,  69,  71,  95,  121 
Peptone,  69,  71,  9s,  119 
ampho,  9S,  120 
anti,  120 

differentiation  of,  from  proteoses,  120 
experiments  on,  120,  121 
in  urine,  323,  337 
tests  for,  337 
separation  of,  from  proteoses,  120 
Periodide  test  for  choline,  273 
Peroxidases,  5,  239 
Pettenkofer's  test  for  bile  acids,  163,  344 

Mylius's  modification   of,    164, 

344 
Neukomm's     modification     of, 
164,  344 
Phenaceturic  acid,  284,  309 
Phenol,  169 

tests  for,  177 
Phenolphthalein  as  indicator,  131,  133 
preparation  of,  133 
test  for  blood  in  feces,  185 
Phenol-sulphuric  acid,  283,  297 
Phenylacetic  acid,  171 
Phenyl- a-amino  propionic  acid,  74,  78 
Phenylalanine,  69,  72,  74,  78 
Phenyldextrosazone,  28 

crystalline  form  of,  Plate  III,  opposite  p.  28 
Phenylethylamine,  171 
Phenylhydrazine,  28,  29 

acetate  solution,  preparation  of,  28 
mixture,  preparation  of,  28 
reaction,  28,  324 

Cipollina's  modification  of,  29,  325 
Phenyllactosazone,  crystalline  form  of,  Platd  111, 

opposite  p.  28 
Phenylmaltosazone,  crystalline  form  of  Plate  III, 

opposite  p.  28 
Phenylpotassium  sulphate,  297 
Phenylpropionic  acid,  171 
Phosphates  in  urine,  276,  284,  317 
detection  of,  320 
experiments  on,  319 
quantitative  determination  of,  413 


INDEX. 


469 


Phosphatase,  3 

Phosphatese,  3 

Phosphatides.  94,  114.  iS9.  27° 

Phosphocamic  acid,  ass,  260,  284,  310 

Phosphoproteins,  94,  9s.  m.  "4 

Phosphorized  compounds  in  urine,  284,  310 

Physiological  constituents  of  urine,  283 

Phj-tasc,  s 

Pigments  of  urine,  274,  284,  310 

Pine  wood  test  for  indole,  176 

Piria's  test  for  tyrosine,  91 

Polariscope,  use  of,  36 

in  detection  of  conjugate  glycuronates,  352 
in  determination  of  dextrose,  36,  332 
^-oxbutyric  acid,  3Si 
Polypeptides,  69,  71,  95 
Polysaccharides,  2s,  47 
classification  of,  25 
properties  of,  47 
Posner's  modification  of  biuret  test,  100 
Potassium  in  urine,  284,  320 

Potassium    indoxyl-sulphate    (see  .Indican,    pp. 
169,  283,  297,  416.) 
formula  for,  169,  298 
origin  of,  168,  297 
tests  for,  298 
Potassium  iodide  test  for  albumin,  105,  336 
Primary  protein  derivatives,  94,  114 
Primary  proteoses,  121 

Products  of  protein  hydrolysis,  69,  72,  73,  74 
Prolamins.  93,  iii 

classification  of,  93 
Proline.  69,  72,  73.  88.  iii,  127,  149 
crystalline  form  of  laevo-o-,  88 
crystalline  form  of  copper  salt  of,  89 
Prosecretin,  148 
Protagon,  268,  269 

preparation  of,  271 
structure  of,  270 
Protamines,  classification  of,  93,  95 
Proteans,  94,  i  is 
Protease,  gastric,  11 

experiments  on,  11 
pancreatic,  ii 

experiments  on.  11 
vegetable,  11 
Proteases,  1 1 

experiments  on.  1 1 
Proteins,  68,  92,  323,  332 

acetic    acid    and    potassium    ferro-cyanide 

test  for,  105 
Acree-Rosenheim  test  on,  100 
action  of  alkaloidal  reagents  on,  104 
action  of  metallic  salts  on,  103 

mineral  acids,  alkalies  and  organic  acids 
on,  103 
Adamkiewicz  reaction  on,  97 
Bardach's  reaction  on,  10 1 
biuret  test  on,  98 
chart  for  use  in  review  of,  1 33 
chemical  composition  of,  68 
classification  of,  93,  9s 
coagulation,  influence  of  salts  upon,  117 
coagulation  or  boiling  test  for,  io6 
color  reactions  of,  97 
conjugated,  94,  95,  112 
decomposition  of,  68,  72,  73,  74 
by  hydrolysis,  69 


Proteins,  decomposition  of,  by  oxidation,  69 
products  of,  69,  72,  73,  74,  76 
experiments  on,  89 
separation  of,  89 
study  of,  69,  72,  89 
derived,  94,  114 
formation  of  fat  from,  142 
formulas  of,  69 
Heller's  ring  test  on,  104 
importance  of,  to  life,  68 
Hopkins-Cole  reaction  on.  98 
in  urine,  323,  332 
test  for,  333 
Liebermann's  reaction  on,  100 
Millon's  reaction  on,  97 
molecular  weights  of,  69 
Posner's  reaction  on,  100 
precipitation  of,  by  alcohol,  107 
alkaloidal  reagents,  104 
metallic  salts,  103 
mineral  acids,  103 
precipitation  reactions  of,  T02 
quantitative  determination  of,  in  milk,  438, 

439 
review  of,  122 
Robert's  ring  test  on,  104 
salts  of,  102 

salting-out  experiments  on,  106 
scheme  for  separation  of,  123 
simple,  93,  95 
synthesis  of,  71,  75 
xanthoproteic  reaction  on,  97 
coagulated,  94,  117 

biuret  test  on,  119 
formation  of,  117 
Hopkins-Cole  reaction  on,  119 
Millon's  reaction  on,  119 
solubility  of,  119 
xanthoproteic  reaction  on,  119 
Protein-coagulated  enzymes,  3,  128,  152,  is6 
Proteins,  conjugated,  94,  112 
classes  of,  94,  112 

experiments  on,  63,  113,  209,  241,  247 
nomenclature  of,  94,  112 
occurrence  of,  112 
Protein-cystine,  8i 
Protein  derivatives,  primary,  70,  94,  114 

secondary,  70,  94,  119 
Proteins  of  milk,  235,  236,  238 

quantitative  determination  of,  438,  439 
Proteolytic  enzymes  (see  Proteases,  p.  ix) 
Proteolysis,  peptic,  127 

tryptic,  128,  149 
Proteose,  69,  94,  9S.  i'9 

v.  Aldor's  method  for  detection  of,  338 
biuret  test  on,  120 
coagulation  test  on,  120 
deutero,  94,  95,  120 
differentiation  of,  from  peptone,  1 20 
experiments  on,  120,  121 
hetero,  95,  120 
in  urine,  3231,  33  7 
test  for,  337 
potassium  ferrocyanide  and  acetic  test  on, 

121 
powder,  preparation  of,  121 
precipitation  of,  by  nitric  acid,  lai 
by  picric  acid,  121 


470 


IXDEX. 


Proteose,   precipitation  of,  by   potassio-mercuric 
iodide,  121 
by  trichloracetic  acid,  1 2 1 
primary,  121 
proto,  94,  95,  121 

Schulte's  method  for  detection  of,  338 
secondary,  121 

separation  of,  from  peptones,  120 
Protoproteose,  94,  95,  121 
Proteoses  and  peptones,  94,  95,  119,  120 
separation  of,  1 20 
tests  on,  121 
Proteose-peptone,  120 

Proteose-peptone,  coagulation  test  on,  120 
experiments  on,  120 
Millon's  reaction  on,  120 
precipitation  of,  by  nitric  acid,  120 
by  picric  acid,  120 
Prothrombin,  203,  204 
Pseudo-globulin,  194,  195 

Ptomaines  and  leucomaines  in  urine,  284,  312 
Ptyalin  (see  Salivary  amylase,  4,  60) 
Purdy's  method  for  determination  of  dextrose,  387 

solution,  preparation  of,  387 
Purine  bases,  113,  284,  312,  428 

in  urine,  quantitative  determination  of,  428 
Pus  casts  in  urinary  sediments,  369,  374 
Pus  cells  in  urinary  sediments,  369 
Putrefaction,  indican  as  an  index  of,  169,  297 
Putrefaction  mixture,  preparation  of  a,  171 
Putrefaction  products,  169 

experiments  on,  171 
most  important,  169 
tests  for,  1  75 
Pyloric  glands,  124 
Pyrocatechin-sulphuric  acid,  283,  297 
a-pyrrolidine-carboxylic  acid  (see  Proline,  pp.  69, 


Quadriurate,  365 

Qualitative  analysis  of  the  products  of  salivary 
digestion,  67 

stomach  contents,  137 
Quantitative  analysis  of  blood,  442 

of  gastric  juice,  440 

of  milk,  435 

of  urine,  383 
Quantitative  determination  of  ammonia  in  urine, 
.599 

amylolytic  activity,  18 

acetone  in  urine,  423 

acetone  and  diacetic  in  urine,  421 

acidity  of  urine,  427 

allanto'in  in  urine,  432 

amino  nitrogen,  404 

ash  of  milk,  438 

caseinogen  of  milk,  438,  439 

catalase,  23 

chlorides  in  urine,  417 

creatine  in  urine,  416 

creatinine,  415 

dextrose  in  urine,  384 

diacetic  acid  in  urine,  425 

fat  in  milk,  435 

fecal  amylase,  189 

fecal  bacteria,  191 

hippuric  acid  in  urine,  406 

indican  in  urine,  416 


Quantitative    determination   of     lactalbumin   in 
milk,    439 

lactose  in  milk,  440 

nitrogen  in  urine,  401 

oxalic  acid  in  urine,  433 

^-oxybutyric  acid  in  urine,  425 

peptic  activity,  19 

phosphorus  in  urine,  413 

protein  in  milk,  438 

protein  in  urine,  383 

purine  bases  in  urine,  428 

purine  nitrogen,  431 

sulphur  in  urine,  408 

total  solids  in  milk,  438 

total  solids  in  urine,  434 

tryptic  activity,  22 

urea  in  urine,  392 

uric  acid  in  urine,  389 
Quevenne  lactometer,   determination   of  specific 
gravity  of  milk  by,  438 

Raflfinose,  25,  47 

Rancid  fat,  141 

Raw  and  heated  milk  tests,  240 

Reaction  of  the  urine,  276,  317 

Reduced  alkali-hcematin,  219 

Reduced  haemoglobin,  216 

Reductases,  239 

Reichert's    method    for    crystallization    of    oxy- 

haemoglobin,  214 
Remont's  method  for  detection  of  salicylic  acid 

and  salicylates,  243 
Rennin,  gastric,  125,  128 

action  of,  upon  caseinogen,  128,  236 

experiments  on,  136,  241 

influence  of,  upon  milk,  128,  236 

in  gastric  juice,  absence  of,  129 

nature  of  action  of,  128,  236 

occurrence  of,  128 
Rennin,  pancreatic,  149,  152 
experiments  on,  157 
Rennin-pepsin  controversy,  129 
Reticulin,  112 

Reversibility  of  enzyme  action,  8,  62 
Reynolds-Gunning  test  for  acetone,  347 
Rhamnase,  s 
Rhamnose,  25,  43 
Ricin,  12,  208 
Riegler's  reaction,  29,  325 
Rigor  mortis,  254 
Ring  test  for  urobilin,  312 

Roaf's  method  for  crystallizing  hippuric  acid,  301 
Robin's  reaction  for  urorosein,  359 
Robert's  ring  test  for  protein,  104,  334 

reagent,  preparation  of,  104,  334 
Rosenheim's  bismuth  test  for  choline,  273 
Rosenheim's  periodide  test  for  choline,  273 
Rose's  method  for  determination  of  pepsin,  21 
Rossi's  reaction  for  indican,  299 
Rothera's  reaction  for  acetone,  347 
Rubner's  test  for  lactose  in  urine,  354 

Saccharide  group,  26 
Saccharose  (see  Sucrose) 
Sahli's  desmoid  reaction,  135 
Saliva,  59 

alkalinity  of,  60 

amount  of,  60 


INDEX. 


471 


Saliva,  bacteria  in,  6.{ 
biuret  test  on,  6.5 
calcium  in,  64 
chlorides  in,  64 
constituents  of,  60 
diKCStion  of  dry  starch  by,  65 
diKestion  of  inulin  by,  65 
digestion  of  starch  paste  by,  61,  65 
dilution  of,  influence  on  digestion,  61 
enzymes  contained  in,  60 
excretion  of  potassium  iodide  in,  67 
inor»;anic  matter  in,  tests  for,  64 
Millon's  reaction  on,  63 
mucin  from,  preparation  of,  63 
nitrites  in,  test  for,  64 
phosphates    in,  test  for,  64 
potassium  thiocyanate  in,  60 
reaction  of,  60,  63 
secretion  of,  59 
specific  gravity  of,  60,  63 
sulphates  in,  test  for,  64 
tests  for.  63 
thiocyanates  in.  60,  64 
tripeptide-splitting  enzymes  in,  62 
Salivary  amylase,  i,  4,  10,  60,  126 

activity  of,  in  stomach,  61,  126 
inhibition  of  activity  of,  61 
nature  of  action  of,  60,  61 
products  of  action  of,  6t 
Salivary  digestion,  59 

influence  of  acids  and  alkalis  on,  61,  66 
dilution  on,  61,  65 
metallic  salts  on,  66 
temperature  on,  65 
nature  of  action  of  acids  and  alkalis  on, 

66 
qualitative  analysis  of  products  of,   67 
Salivary  digestion  in  stomach,  61,  126 
Salivary  glands.  59 
Salivary  stimuli,  59 

Salkowski-Autenrieth-Barth    method    for    deter- 
mination of  oxalic  acid  in  urine.  433 
Salkowski's  method  for  determination  of  purine 

bases.  430 
Salkowski-Schippers  reaction  for  bile  pigments, 

■  63,  34,i 
Salkowski's  test  for  cholesterol,  166,  272 

for  creatinine,  297 
Salmine,  85,  86,  72,  73,  93,  95 
Saponification,  140,  144 

of  lard,  146 
Salted  plasma,  preparation  of,  214 
Salting-out  experiments  on  proteins,  103,  106 
Sarcolactic  acid,  256 

Scallops,  preparation  of  glycogen  from,  263 
Schalfijew's   method   for  preparation   of   ha;niin, 

212 

Schema  for  "blood  counting,"  232 
Scheme  for  analysis  of  biliary  calculi,  165 

bone  ash,  253 

stomach  contents,  138 

urinary  calculi.  381 
separation  of  carbohydrates,  58 

of  proteins,  1 23 
Scherer's  coagulation   method  for  determination 

of  albumin  in  urine,  383 
Schiffs  reaction  for  cholesterol,  166,  272 

for  uric  acid,  293 


Schifl's  reagent,  preparation  of,  166,  272 
Schmidt's  nuclei  test  for  pancreatic  insufficiency, 

188 
Schmidt's  test  for  hydrobilirubin,   186 
Schulte's    method    for   detection    of    proteose    in 

urine,  338 
Schumm's  modification  of  the  guaiac  test,  209 
Schutz's  law,  statement  of,  8,  20 
Schweitzer's  reagent,  action  of,  on  cellulose,  S4 

preparation  of,  54 
Scleroproteins,  95  (see  Albuminoids) 
Scombrine,  72,  93 
Scombrone,  93,  95 
Scybala,  56,  180 

Secondary  protein  derivatives,  70,  94,  119 
Secondary  proteoses,  1 2 1 
Secretin,  148 
Seliwanoff's  reaction,  40,  356 

reagent,  preparation  of,  40.  356 
Separation  of  feces,   importance  of,  in  nutrition 

and  metabolism  experiments,  180,  189 
Serine,  69,  72,  74,  78 

crystalline  form  of,  78 
formula  for,  78 
Seromucoid,  94,  113,  196 
Serum  albumin,  93,  95,  194.  3^3,  33^ 
in  urine.  323,  332 
test  for,  333 
Serum  globulin.  93,  194,  323,  332 
in  urine,  323,  336 
test  for,  336 
Shackell's  method  for  vacuum  desiccation,  434 
Shaffer's     method    for   determination    of    jJ-oxy- 

butyric  acid,  425 
Sherman's   compressed    oxygen    method    for   de- 
termination of  total  sulphur  in  urine,  4 1  2 
Sherrington's  solution,  preparation  of,   225 
Silicates  in  urine,  284,  322 
Skatole,  169,  171,  176,  179 

tests  for,  I  76 
Skatole-carbonic  acid,  174 

test  for.  177 
Smith's  test  for  bile  pigments.  163,  343 
Soap, salting-out  of ,  145 
Sodium  and  potassium  in  urine,  284,  320 
Sodium    alizarin    sulphonate    as   indicator,    131, 
133 
preparation  of.  133 
Sodium  chloride,  crystalline  form,  213 
Sodium  chloride  in  urine,  284,  316,  417 
Sodium  hydroxide  and  potassium  nitrate  fusion 
method  for  determination  of  total  sulphur  and 
phosphorus  in  urine.  412,  414 
Sodium  hypobromite  solution,  preparation  of,  392 
Sodium  sulphide  solution,  preparation  of,  429 
Solera's  reaction  for  detection  of  thiocyanate  in 
saliva,  64 
test  paper,  preparation  of,  64 
Soluble  starch,  10,  48,  61 
Soxhlet  apparatus  for  extraction  of  fat,  437 
Soxhlet    lactometer,    determination    of    specific 

gravity  of  milk  by,  435 
Specificity  of  enzyme  action,  7 
Spectroscope,  use  of  in  detection  of  blood.  215 
Spermatozoa  in  urinary  sediments.   369.  376 

microscopical  appearance  of  human.  376 
Spiegler's  ring  test  for  protein.  104,  334 
reagent,  preparation  of,  104,  334 


472 


INDEX. 


Spongin,  74 

Sprigg's    method    for    determination    of    peptic 

activity,  19 
Standard  ammonium  thiocyanate  solution,  prepa- 
ration of,  420 
silver   nitrate   solution,    preparation   of,  419 
uranium  acetate  solution,  preparation  of,  413 
Starch,  26,  48 

action  of  alcohol  on  iodide  of,  50 
action  of  alkali  on  iodide  of,  50 

heat  on  iodide  of,  50 
dry,    digestion   of,    by   pancreatic   amylase, 

isi,  156 
dry,  digestion  of,  by  salivary  amylase,  65 
experiments  on,  48 
iodine  test  for,  $0 
microscopical  characteristics  of,  48 
microscopical  examination  of,  48 
potato,  preparation  of,  48 
soluble,  10,  48,  6i 
solubility  of,  48 
various  forms  of,  49 
Starch  group,  26 

Starch  paste,  action  of  tannic  acid  on,  50 
diffusibility  of,  50 

digestion  of,  by  pancreatic  amylase,  150, 
iSS 
by  salivary  amylase,  61,  65 
Fehling's  test  on,  50 
hydrolysis  of,  50 
iodic  acid  paper,  64 
preparation  of,  50 
Steapsin  (see  Pancreatic  lipase,  4,  140,  150,  157) 
Stearic  acid,  269 
Stearin,  140,  23  s 
Stellar  phosphate,  242,  362,  364 
Stercobilin,  179 
Stokes'  reagent,  action  of,  216 

preparation  of,  216 
Stomach,  motor  and  functional  activities  of,  136 
Stomach  contents,  lactic  acid  in  tests  for,  136 
peptide-splitting  enzyme  in,  128 
qualitative  analysis  of,  137 
Stone-cystine,  81 
Sturine,  72,  73,  93 
Sublingual  glands,  characteristics  of  saliva  secreted 

by,  59 
Submaxillary    glands,    characteristics    of    saliva 

secreted  by,  59 
Substrate,  2,  47 
Succinic  acid,  171 
Sucrase,  5,  13,  152 

experiments  on,  13 
vegetable,  13 
Sucrose,  25,  46 

experiments  on,  46 
inversion  of,  46 
production  of  alcohol  from,  47 
structure  of,  46 
Sulphanilic  acid,  359 
Sulphates  in  saliva,  test  for,  64 
Sulphates  in  urine,  284,  315 
experiments  on,  315 
ethereal,  283,  297 

quantitative  determination  of,  409 
inorganic,  284,  314 

quantitative  determination  of,  409 
total,  quantitative  determination  of,  408 


Sulphocyanides  (see  Thiocyanates,  60,  64,  420) 
Sulphur  in  protein,  68,  108 

loosely  combined,  tests  for,  108 

in  urine,  quantitative  determination  of,  408 

acid,  108 

lead  blackening,  108 

mercaptan,  108 

neutral,  283,  303 

oxidized,  108,  109 

unoxidized,  108 
Suspension  of  manganese  dioxide,  430 
Synaptase  (see  Emulsin,  4) 

Tallow  bayberry,  saponification  of,  144 
Tallquist's    haemoglobin    scale,  determination    of 

haemoglobin  by,  224 
Tannic  acid,  influence  of,  on  dextrin,  S3 

on  starch,  50 
Tannin  test  for  carbon  monoxide  hasmoglobin, 

217 
Tanret's  reagent,  preparation  of,  105,  33s 
Tanret's  test,  105,  33S 
Tartar,  formation  of,  60 
Taurine,  159,  166,  255,  260,  283,  303 
derivatives,  283,  303 
formula  for,  159,  260 
preparation  of,  166 
Taurocholic  acid,  159 

group,  1 59 
Taylor's  test  for  acetone,  347 
Teichmann's     crystals,     form     of     (see     Haemin 
crystals,  p.  211) 
test,  210,  340 
Tendomucoid,  94,  112,  113,  247 
biuret  test  on,  247 
chemical  composition  of,  113 
hydrolysis  of,  247 

loosely  combined  sulphur  in,  test  for,  247 
preparation  of,  247 
solubility  of,  247 
Tetrapeptides,  71,  95 
Tetramethylene-diamine,  171 
Thiocyanates  in  saliva,  significance  of,  60 
ferric  chloride  test  for,  64 
Solera's  reaction  for,  64 
Thiocyanates  in  urine,  280,  303 
Thiophene  reaction,  137 
Thoma-Zeiss  haemocytometer,  224 
Thrombin,  5,  203 
Thromboplastine,  204 
Thymus  histone,  93 
Thymol,  formula  for,  281 

interference  of,  in  Lieben's  acetone  test,  347 
interference  in  Heller's  ring  test,  333 
use  of,  as  preservative,  281 
Tincture  of  iodine,  preparation  of,  453 
Tissue,  adipose,  experiments  on,  128,  253 
conne«tive,  245 

■white  fibrous,  246 

composition  of,  246 
experiments  on,  247 
yellow  elastic,  249 

composition  of,  249 
experiments  on,  249 
epithelial,  245 

experiments  on,  245 
muscular,  254 

experiments  on,  261 


INDEX. 


473 


Tissue,  nervous,  268 

experiments  on,  271 
osseous,  2  5  I 

experiments  on,  252 
Tissue  debris  in  urinary  sediments,  369,  378 
Titanium  tetrachloride  as  cellulose  solvent,  53 
Toison's  solution,  preparation  of,  225 
ToUen's  reaction  on  conjugate  glycuronates,  352 
arabinose,  4  a 
galactose,  41 
pentoses  in  uripe,  353 
Topfer's    method    for    quantitative    analysis    of 

gastric  juice,  440 
Topfer's  reagent,  as  indicator,  131,  132 

preparation  of,  132 
Total  solids,  of  milk,  quantitative  determination 
of,  438 
of    urine,    quantitative    determination 
of,  434 
Total  sulphur  of  urine,  quantitative  determination 
of,  409-413 
phosphorus    of    urine,    quantitative    deter- 
mination of,  414 
Trehalase,  s 
Trichloracetic  acid,  precipitation  of  protein  by, 

104 
Trimethyl-oxyethyl-ammonium    hydroxide     (see 

Choline,  269) 
Trioses,  25 
Tripeptides,  71,  95 
Triple  phosphate,  277,  319,  362,  380 

crystalline  form  of,  319  ; 

formation  of,  319 
Trisaccharides,  2s,  47 
Trommer's  test,  31,  326 
Tropaeolin  00,  as  indicator,  131,  132 

preparation  of,  132 
Trj-psin  (see  also  Pancreatic  protease,  5,  11,  22, 
128,  149,  154 
action  of,  upon  proteins,  69,  128,  149,  154 
experiments  on,  is4 
influence  of  alkalis  and  mineral  acids  upon, 

149 
nature  of,  149 
pure,  preparation  of,  149 
Trypsinogen,  4,  6,  149 

activation  of,  6,  149 
Tryptic  digestion,  128,  149 

influence  of  bile  on,  155 

metallic  salts  on,  154 
most  favorable  reaction  for,  154 

temperature  for,  154 
products  of,  149,  153 
Tryptic  proteolysis,  128,  149 
Tryptophane,  is,  69,  72,  73,  82,  149,  154 
bromine  water  test  for,  is4 
formula  for,  82 

group  in  the  protein  molecule,  97,  98 
Hopkins-Cole  reaction  for,  98 
occurrence  of,  as  a  decomposition  product  of 

protein,  69,  72,  73,  82, 
occurrence   of,   as   an   end-product   of   pan- 
creatic digestion,  149,  154 
Tussah  silk  fibroin,  73 

"Twinning"  of  oxyhemoglobin  crystals,  202 
Tyrosinase,  5 

Tyrosine,  s.  69i  72,  74i  79,  90i  97.  I27>  149,  362,  367 
crystalline  form  of,  8  r 


Tyrosine,  experiments  on,  90 

formula  for,  79 

Hoffmann's  reaction  for,  91 

in  urinary  sediments,  362,  367 

microscopical  examination  of,  90 

Morncr's  test  for,  91 

occurrence  of,  69,  72,  74,  79,  149 

Piria's  test  for,  91 

salts  of,  79 

separation  of,  from  leucine,  90 

solubility  of,  91 

sublimation  of,  91 
Tyrosine-sulphuric  acid,  91 


V.  Udrdnsky's  test  for  bile  acids,  164,  344 
Uflelmann's  reagent,  preparation  of,  137 

reaction  for  lactic  acid,  136 
Unknown  substances  in  urine,  323,  359 
Unorganized  ferments,  i 

sediments  in  urine,  361,  362 
Uranium   acetate   method   for   determination   of 

total  phosphates  in  urine,  413 
Uracil,  113 

Wheeler- Johnson  reaction  for,  113 
Urate,  ammonium,  crystalline  form  of,  Plate  VI, 
opposite  p.  365 
sodium,  crystalline  form  of,  366 
Urates  in  urinary  sediments,  362,  365 
Urea,  196,  255,  283,  284 

crystalline  form  of,  284 

decomposition  of,  by  sodium  hypobromite, 

286,  289 
excretion  of,  28s,  287 
experiments  on,  287 
formation  of,  286 
formula  for,  284 
furfurol  test  for,  290 
isolation  of,  from  the  urine,  287 
melting-point  of,  288 
quantitative  determination  of,  392-399 
Urea  nitrate,  287,  289 

crystalline  form  of,  287 
formula  for,  287 
oxalate,  287,  289 

crystalline  form  of,  289 
formula  for,  287 
Urease,  s 

Urethral  filaments  in  urinary  sediments,  369,  377 
Uric  acid,  32,  196,  255,  277,  283,  290,  362,  364, 
389 
crystalline  form  of,  pure,  293 
endogenous,  291 
exogenous,  291 
experiments  on,  292 
formula  for,  290 

Oanassini'e  test,  293 
in  leukaemia,  293 
in  urinary  sediments,  362,  364 

crystalline  form  of  Plate  V,  oppo- 
site p.  291,  36s 
isolation  of,  from  the  urine,  292 
Moreigne's  reaction  for,  293 
murexide  test  for,  292 
origin  of,  291 
quantitative  determination  of,  389 

Folin-Schaffer  method  for,  389 
Heintz  method  for,  390 


474 


INDEX. 


Uric  acid,  quantitative  determination  of,  Kriiger 
and  Schmidt's  method  for,  391 
reducing  power  of,  32,  293,  327 
Schiff's  reaction  for,  293 
Uricase,  5,  16 

Uricolytic  enzymes,  3,  5,  16 
experiments  on,  16 
Urinary  calculi,  379 

calcium  carbonate  in,  380 
oxalate  in,  380 
cholesterol  in,  382 
compound,  379 
cystine  in,  380 
fibrin  in,  380 
indigo  in,  382 
phosphates  in,  380 
scheme  for  chemical  analysis  of,  381 
simple,  379 

uric  acid  and  urates  in,  380 
urostealiths  in,  380 
xanthine  in,  380 
Urinary  concrements  (see  Urinary  calculi,  p.  379) 
Urinary  concretions  (See  Urinary  calculi,  p.  379) 
Urinary  sediments,  361 

ammonium    magnesium    phosphate    in, 

362 
animal  parasites  in,  369,  378 
calcium  carbonate  in,  362,  363 
oxalate  in,  362 

calcium  phosphate  in,  362,364 
sulphate  in,  362,  364 
casts  in,  369,  371 
cholesterol  in,  362,  366 
collection  of,  361 
cylindroids  in,  369,  376 
cystine  in,  362,  366 
epithelial  cells  in,  369 
erythrocytes  in,  369,  376 
fibrin  in,  369,  378 
foreign  substances  in,  369,  378 
haematoidin  and  bilirubin  in,  362,  367 
hippuric  acid  in,  362,  367 
indigo  in,  362,  368 
leucine  and  tyrosine  in,  362,  367 
magnesium  phosphate  in,  362,  368 
melanin  in,  362,  368 
micro-organisms  in,  369,  378 
organized,  361,  369 
pus  cells  in,  369 
spermatozoa  in,  369,  376 
tissue  ddVjris  in,  369,  378 
unorganized,  361,  362 
urates  in,  362,  36."; 
urethral  filaments  in,  369,  377 
uric  acid  in,  362,  364 
xanthine  in,  362,  368 
Urination,  frequency  of,  276 
Urine,  274-434 

acetone  in,  323,  34s 

acidity  of,  276,  31  7 

acid  fermentation  of,  277 

albumin  in,  323,  332 

alkaline  fermentation  of,  276 

alantoin  in,  283,  303 

amino  acids,  283 

ammonia  in,  276,  284,  313,  399 

aromatic  oxyacids  in,  283,  306 

benzoic  acid  in,  283,  307 


Urine,  bile  in,  323,  342 

blood  in,  323,  339 

calciuna  in,  284,  320 

carbonates  in,  284,  321 

chlorides  in,  284,  316 

collection  of,  281 

conjugate  glycuronates  in,  323,  351 

color  of,  274 

creatine,  258,  283,  323 

creatinine  in,  283,  294 

dextrose  in,  323 

diacetic  acid  in,  323,  348 

electrical  conductivity  of,  281 

enzymes  in,  284,  309 

ethereal  sulphuric  acid  in,  283,  297 

fat  in,  323,  353 

fluorides  in,  284,  322 

freezing-point  of,  279 

galactose  in,  323,  355 

general  characteristics  of,  274 

globulin  in,  323,  336 

Haser's  coefficient  for  solids  in,  279,  434 

hasmatoporphyrin  in,  323,  353 

hippuric  acid  in,  283,  300,  367 

hydrogen  peroxide  in,  284,  322 

inorganic  physiological  constituents  of,  28 
313 

inosite  in,  323,  357 

iron  in,  284,  321 

lactose  in,  323,  354 

laevulose  in,  323,  355 

laiose  in,  323,  358 

leucomaines  in,  284,  312 

Long's  coefficient  for  solids  in,  27S,  434 

magnesium,  in,  284,  320 

melanin  in,  323,  358 

neutral  sulphur  compounds  in,  283,  303 

nitrates  in,  284,  322 

nucleoprotein  in,  283,  308,  323,  339 

odor  of,  27s 

organic  physiological  constituents  of,   283 

oxalic  acid  in,  283,  302 

oxaluric  acid  in,  283,  308 

/3-oxybutyric  acid  in,  323,  349,  425 
pathological  constituents  of,  323 
paralactic  acid  in,  256,  284,  309 
pentoses  in,  323,  352 
peptone  in,  323,  337 
phenaceturic  acid  in,  284,  309 
phosphates  in,  284,  317 
phosphorized  compounds  in,  284,  310 
physiological  constituents  of,  283 
Ijigments  of,  274,  284,  310 
potassium  in,  284,  320 
proteins  in,  323,  352 
proteoses  in,  323,  332,  337 
ptomaines  in,  284,  312 
purine  bases  in,  284,  312,  428 
quantitative  analysis  of,  383-434 
reaction  of,  276,  317 
silicates  in,  284,  322 
sodium,  in,  284,  320 
solids  of,  278,  434 
specific  gravity  of,  278 
sulphates  in,  284,  314,  408 
transparency  of,  275 
unknown  substances  in,  323,  359 
urea  in,  283,  284 


INDEX. 


475 


Urine,   uric  acid  in,  2.S.},  jgo 

urorosein  in,  ^j.i,  3sH 

volatile  fatty  acids  in,  284. ,509 

volume  of,  274 
Urobilin,  274,  284,  .?'o 

tests  for,  i  1 1 
Urochrome,  274,  284,  310,  330 
Uroerythrin,  274,  284,  3>o.  330 
I'roferric  acid,  283,  303,  3S9 
I'roleucic  acid,  283,  306 
Urorosein,  323,  358^ 

tests  for,  3  59 

Valine,  69,  72,  74,  83 

Van  Slyke's  method  for  determination  of  amino 

nitrogen,  408 
Vegetable  amylase,  4,10 
lipase,  4,12 
protease,  1 1 
sucrase,  13 
Vegetable  globulins,  93,  95,  109 
Vegetable  gums,  26 
With     lactometer,     determination     of     specific 

gravity  of  milk  by,  43  5 
Viscosity  test,  64 
Vitellin,  94,  95 

Volatile  fatty  acids,  169,  172,  284.  309 
Volhard-Amold    method    for    determination    of 

chlorides,  4 1 9 
Volhard-Harvey    method    for    determination    of 

chlorides,  420 
Volume  of  the  urine,  274 

Water  at  meals,  influence  of,  61,  62,  124.  151,  180, 
182,  183,  314 

softened,  62 
Wax  myrtle,  144 

Waxy  casts  in  urinary  sediments,  369,  373 
Weber's  guaiac  test  for  blood  in  feces,  186 
Weinland,  formation  of  fat  from  protein,  T43 
Welker's  electrical  bath,  397 

modified  method  for  purine  bases,  428 
Weyls  test  for  creatinine,  296 


Wheeler-Johnson  reaction  for  uracil  and  cytosine, 

113 
White  fibrous  connective  tissue,  246 

experiments  on,  247 
Wiechowski's     method     for     determination     of 

allantoin,  304,  432 
Wilkinson  and  Peters'  test,  240 
Wirsing's  test  for  urobilin,  31  1 
Witche's  milk,  237 

Wohlgemuths'    method    for    quantitative    deter- 
mination of  amylolytic  activity,  18 
Author's  modification  of,  189 

Xanthine,  255,  258,  261,  284,  312 

crystalline  form  of,  258 

formula  for,  261 

in  urinary  sediments,  362,  368 

isolation  of,  from  meat  extract,  266 

Weidel's  reaction  for,  266 

bases  (see    Purine  bases,  pp.   113,   284,   312, 
428) 
Xanthine  silver  nitrate,  265,  266 

crystalline  form  of,  266 
test,  266 
Xanthoproteic  reaction,  97 
Xantho-oxidase,  5 
Xylose,  25,  43 

orcinol  reaction  on,  43 

phenylhydrazine  reaction  on.  43 

Tollens'  reaction  on,  43 

Yellow  elastic  connective  tissue,  249 
composition  of,  249 
experiments  on,  249 

Zappert  slide,  227 
Zein,  72,  74,  93,  95,  112 

decomposition  of,  72,  74 
Zeller's  test  for  melanin,  358 
v.  Zeynek  and  Nencki's  haemin  test,  21  2,  340 
Zikel  pektoscope,  279 
Zymase,  classification  of,  5 

preparation  of,  2 
Zymo-exciter,  6 
Zymogen,  6,  127, 


